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The spread of multidrug antibiotic resistance (MDR) is a widely recognized crisis in the treatment of bacterial infections, including those occurring in military communities. Recently, the World Health Organization published its first ever list of antibiotic-resistant "priority pathogens" - a catalog of 12 families of bacteria that pose the greatest threat to human health with A. baumannii listed in the "Priority 1: Critical" category of pathogens. With the increasing prevalence of antibiotic resistance and limited development of new classes of antibiotics, alternative antimicrobial therapies are needed, with lytic bacteriophage (phage) specifically targeted against each of the high priority bacterial infections as a potential approach currently in development toward regulatory approval for clinical use. Balb/c mice were prophylactically administered PBS or phage selected against A. baumannii strain AB5075. After 3 weeks, mice were anesthetized, wounded (dorsal), and challenged topically with AB5075. Following infection, mice were subsequently treated with PBS or phage for three consecutive days, and evaluated for 3 weeks to assess the safety and efficacy of the phage treatment relative to the control. We assessed mortality, bacterial burden, time to wound closure, systemic and local cytokine profiles, alterations in host cellular immunity, and finally presence of neutralizing antibodies to the phage mixture. In our study, we found that prophylactic phage administration led to a significant reduction in monocyte-related cytokines in serum compared to mice given PBS. However, we detected no significant changes to circulating blood populations or immune cell populations of secondary lymphoid organs compared to PBS-treated mice. Following prophylactic phage administration, we detected a marked increase in total immunoglobulins in serum, particularly IgG2a and IgG2b. Furthermore, we determined that these antibodies were able to specifically target phage and effectively neutralize their ability to lyse their respective target. In regards to their therapeutic efficacy, administration of phage treatment effectively decreased wound size of mice infected with AB5075 without adverse effects. In conclusion, our data demonstrate that phage can serve as a safe and effective novel therapeutic agent against A. baumannii without adverse reactions to the host and pre-exposure to phage does not seem to adversely affect therapeutic efficacy. This study is an important proof of concept to support the efforts to develop phage as a novel therapeutic product for treatment of complex bacterial wound infections.

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Staphylococcus aureus-associated infections can be difficult to treat due to multidrug resistance. Thus, infection prevention is critical. Cationic antiseptics, such as chlorhexidine (CHX) and benzalkonium chloride (BKC), are liberally used in health care and community settings to prevent infection. However, increased administration of antiseptics has selected for S. aureus strains that show reduced susceptibilities to cationic antiseptics. This increased resistance has been associated with carriage of specific efflux pumps (QacA, QacC, and NorA). Since prior published studies focused on different strains and on strains carrying only a single efflux gene, the relative importance of these various systems to antiseptic resistance is difficult to ascertain. To overcome this, we engineered a collection of isogenic S. aureus strains that harbored norA, qacA, and qacC, individually or in combination. MIC assays showed that qacA was associated with increased resistance to CHX, cetrimide (CT), and BKC, qacC was associated with resistance to CT and BKC, and norA was necessary for basal-level resistance to the majority of tested antiseptics. When all three pumps were present in a single strain, an additive effect was observed in the MIC for CT. Transcriptional analysis revealed that expression of qacA and norA was significantly induced following exposure to BKC. Alarmingly, in a strain carrying qacA and norA, preexposure to BKC increased CHX tolerance. Overall, our results reveal increased antiseptic resistance in strains carrying multiple efflux pumps and indicate that preexposure to BKC, which is found in numerous daily-use products, can increase CHX tolerance.IMPORTANCES. aureus remains a significant cause of disease within hospitals and communities. To reduce the burden of S. aureus infections, antiseptics are ubiquitously used in our daily lives. Furthermore, many antiseptic compounds are dual purpose and are found in household products. The increased abundance of antiseptic compounds has selected for S. aureus strains that carry efflux pumps that increase resistance to antiseptic compounds; however, the effect of carrying multiple pumps within S. aureus is unclear. We demonstrated that an isogenic strain carrying multiple efflux pumps had an additive resistance phenotype to cetrimide. Moreover, in a strain carrying qacA and norA, increased chlorhexidine tolerance was observed after the strain was preexposed to subinhibitory concentrations of a different common-use antiseptic. Taken together, our findings demonstrate cooperation between antiseptic resistance efflux pumps and suggest that their protective phenotype may be exacerbated by priming with subinhibitory concentrations of household antiseptics.

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Introduction. Severely burned patients are susceptible to bacterial infection within their burn wounds, which frequently leads to sepsis, multiple organ failure and death. The opportunistic pathogen Pseudomonas aeruginosa, an organism inherently resistant to multiple antibiotics, is a common cause of sepsis in these patients.Aim. Development of a topical treatment unrelated to conventional antibiotics is essential for prevention of P. aeruginosa infection and sepsis, leading to a role for the direct application of probiotics or their by-products.Methodology. We examined the effectiveness of 20× concentrated supernatant from Lactobacillus gasseri strain 63 AM (LgCS) grown in de Man, Rogosa and Sharpe broth in inhibiting P. aeruginosa biofilms in vitro, as well as in reducing wound bioburden and P. aeruginosa sepsis in vivo.Results. LgCS inhibited the growth of P. aeruginosa strain PAO1, prevented its biofilm development and eliminated partially developed PAO1 biofilms. In the murine model of thermal injury, a single injection of LgCS following injury and PAO1 infection reduced mortality to 0 % and prevented systemic spread (sepsis). Furthermore, a second injection of LgCS 24 h after the first eliminated PAO1 from the wound. In the murine dorsal excision infection model, either LgCS or ceftazidime treatment of the PAO1-infected wound significantly reduced the mortality rate among infected mice, while combining LgCS with ceftazidime eliminated mortality.Conclusion. These results suggest the potential of LgCS in preventing sepsis from P. aeruginosa infection in severely burned and other immunocompromised patients.

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