Bacteriophages targetingAcinetobacter baumanniicapsule induce antimicrobial resensitization

Abstract

21Carbapenem-resistant Acinetobacter baumannii is responsible for frequent, hard-to-treat and often fatal 22 healthcare-associated infections. Phage therapy, the use of viruses that infect and kill bacteria, is an approach 23 gaining significant clinical interest to combat antibiotic-resistant infections. However, a major limitation is that 24 bacteria can develop resistance against phages. Here, we isolated phages with activity against a panel of A.25 baumannii strains and focused on clinical isolates AB900 and A9844 and their phages for detailed 26 characterization. As expected, coincubation of the phages with their hosts in vitro resulted in the emergence of 27 phage-resistant bacterial mutants. Genome sequence analysis revealed that phage-resistant mutants harbored 28 loss-of-function mutations in genes from the K locus, responsible for the biosynthesis of the bacterial capsule. 29Using molecular biology techniques, phage adsorption assays, and quantitative evaluation of capsule 30 production, we established that the bacterial capsule serves as the primary receptor for these phages. As a 31 collateral phenotype of impaired capsule production, the phage-resistant strains could not form biofilms, became 32 fully sensitized to the human complement system, showed increased susceptibility to beta-lactam antibiotics, 33 and became vulnerable to additional phages. Finally, in a murine model of bacteremia, the phage-resistant A. 34 baumannii demonstrated a diminished capacity to colonize blood and solid tissues. This study demonstrates 35 that phages can be used not only for their lytic activity but, if combined with a posteriori knowledge of their 36 receptors and the mechanism of bacterial resistance, for their potential synergy with other antimicrobial agents, 37 thus providing even broader clinical options for phage therapy. 39 Keywords 40Phage therapy, Acinetobacter baumannii, bacterial capsule, phage receptors, antimicrobial resistance. 41 42 2 Introduction 43In 2019, antimicrobial resistance was listed by the World Health Organization (WHO) as one of the top 44 ten threats to global health (1). Multidrug resistant (MDR) infections are consistently associated with poor clinical 45 outcomes and represent a significant financial burden on the healthcare system (2, 3). Also in 2019, the WHO 46 and Centers for Disease Control and Prevention (CDC) prioritized carbapenem-resistant Acinetobacter 47 baumannii as a pathogen critically requiring research and development of new antimicrobial strategies (4, 5). 48A. baumannii, a gram-negative coccobacillus, is a member of the ESKAPE group of pathogens, which are 49 prominent for causing frequent and hard-to-treat healthcare-associated infections (6). 50As a species, A. baumannii is highly resilient, and capable of surviving for months in biofilms on abiotic 51 surfaces (7, 8). It can cause pneumonia, bacteremia, urinary tract infections, meningitis and wound infections, 52 particularly in the context of intensive care units, and is frequently associated with indwelling med...

High-throughput mapping of the phage resistance landscape in E. coli

“…In particular, identification of phages that differ in their receptor use or against which cross-resistance is unlikely to evolve would allow for better design of such therapies [ 23 , 37 , 242 ]. Moreover, identifying phages that select for resistance that have interrelated phenotypic consequences with, for example, antibiotic sensitivity is a recent advancement in the field that could directly benefit from these screening approaches [ 33 , 53 , 97 , 243 ]. By combining fitness datasets for phages and antibiotics or phage-antibiotic combination therapies [ 244 – 246 ], such screens could provide an avenue for performing high-throughput search for genetic trade-offs or “evolutionary traps” [ 33 , 53 , 97 , 243 ] that could provide a much-needed solution to overcome the antibiotic-resistance pandemic.…”

“…Despite nearly a century of pioneering molecular work, the mechanistic insights into phage specificity for a given host, infection pathways, and the breadth of bacterial responses to different phages have largely focused on a handful of individual bacterium-phage systems [9][10][11][12][13]. Bacterial sensitivity/resistance to phages is typically characterized using phenotypic methods such as crossinfection patterns against a panel of phages [14][15][16][17][18][19][20][21][22][23][24][25][26][27] or by whole-genome sequencing of phageresistant mutants [28][29][30][31][32][33]. As such, our understanding of bacterial resistance mechanisms against phages remains limited, and the field is therefore in need of improved methods to characterize phage-host interactions, determine the generality and diversity of phage resistance mechanisms in nature, and identify the degree of specificity for each bacterial resistance mechanism across diverse phage types [13,25,26,[34][35][36][37][38][39][40][41][42][43][44][45][46][47]…”

Multidrug-resistant Acinetobacter baumannii as an emerging concern in hospitals

“…Furthermore, they found that these mutants showed a decrease in biofilm formation antibiotic resistance. This study suggested that these bacteriophages cannot be used for their lytic activity only but the combination between understanding of phage receptors and bacterial resistance mechanism provides the best knowledge of the potential synergy effect of both phage and antimicrobial agents [ 91 , 114 ]. Therefore, future attention should be focused on bacteriophage therapy especially for A. baumannii with MDR resistance, which causes many epidemics in hospitals.…”

Biological Control of Acinetobacter baumannii: In Vitro and In Vivo Activity, Limitations, and Combination Therapies

“…While bacteriophage-based therapies hold immense potential as biological control agents; the emergence of bacteriophage resistance mediated via: (1) receptor adaptations (mutations of phenotypical alteration resulting in decreased bacteriophage adsorption); (2) host defence systems (molecular pathways preventing or suppressing phage infections); and (3) phage-derived defence systems (molecular pathways facilitating bacterial competition of host), remains a major obstacle, hampering the effective application of this treatment ( Figure 4 ) (extensively reviewed by Egido et al [ 98 ]). The underlying resistance mechanisms have subsequently been identified and exploited in an approach referred to as “bacteriophage steering”, which involves the “exploitation-specific fitness trade-offs” associated with bacteriophage resistance, including antimicrobial re-sensitisation, reduced virulence, and colonisation defects [ 106 , 107 ]. For example, Altamirano et al [ 106 ] isolated a Myoviridae bacteriophage (ɸFG02) and an Ackermannviridae bacteriophage (ɸCO01).…”

“…The underlying resistance mechanisms have subsequently been identified and exploited in an approach referred to as “bacteriophage steering”, which involves the “exploitation-specific fitness trade-offs” associated with bacteriophage resistance, including antimicrobial re-sensitisation, reduced virulence, and colonisation defects [ 106 , 107 ]. For example, Altamirano et al [ 106 ] isolated a Myoviridae bacteriophage (ɸFG02) and an Ackermannviridae bacteriophage (ɸCO01). Following co-culturing with the respective A. baumannii host strains, bacteriophage resistance was observed, which correlated with the loss of the CPS.…”