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

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.

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A new class of cell wall-recycling L,D-carboxypeptidase determines β-lactam susceptibility and morphogenesis in Acinetobacter baumannii

10.1101/2021.09.22.461454 ◽

2021 ◽

Author(s):

Yunfei Dai ◽

Victor Pinedo ◽

Amy Y Tang ◽

Felipe Cava ◽

Edward Geisinger

Keyword(s):

Cell Wall ◽

Acinetobacter Baumannii ◽

Cell Envelope ◽

Membrane Lipid ◽

Model Organisms ◽

Complex Cell ◽

Intrinsic Resistance ◽

New Class ◽

Hospital Acquired ◽

Cell Wall Recycling

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 cell elongation 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, elongation, 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-crosslink formation, cell division, and outer membrane lipid homoeostasis, reflecting its pleiotropic influence on cell 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.

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Complete Genome Sequences of Four Extensively Drug-Resistant Acinetobacter baumannii Isolates from Thailand

Microbiology Resource Announcements ◽

10.1128/mra.00949-20 ◽

2020 ◽

Vol 9 (40) ◽

Author(s):

Peechanika Chopjitt ◽

Thidathip Wongsurawat ◽

Piroon Jenjaroenpun ◽

Parichart Boueroy ◽

Rujirat Hatrongjit ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Complete Genome ◽

Sequence Type ◽

Clinical Isolates ◽

Drug Resistant ◽

Genome Sequences ◽

Content Type ◽

Type Isolate ◽

Resistant Genes ◽

Extensively Drug Resistant

ABSTRACT Here, we report the complete genome sequences of four clinical isolates of extensively drug-resistant Acinetobacter baumannii (XDRAB), isolated in Thailand. These results revealed multiple antimicrobial-resistant genes, each involving two sequence type 16 (ST16) isolates, ST2, and a novel sequence type isolate, ST1479.

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Lytic Myophage Abp53 Encodes Several Proteins Similar to Those Encoded by Host Acinetobacter baumannii and Phage phiKO2

Applied and Environmental Microbiology ◽

10.1128/aem.05116-11 ◽

2011 ◽

Vol 77 (19) ◽

pp. 6755-6762 ◽

Cited By ~ 14

Author(s):

Chia-Ni Lee ◽

Tsai-Tien Tseng ◽

Juey-Wen Lin ◽

Yung-Chieh Fu ◽

Shu-Fen Weng ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Burst Size ◽

Sputum Sample ◽

Polyacrylamide Gel Electrophoresis ◽

Multiple Drug ◽

Lytic Phage ◽

Tail Fiber ◽

Drug Resistant ◽

Content Type ◽

Multiple Drug Resistant

ABSTRACTAcinetobacter baumanniiis an important Gram-negative opportunistic pathogen causing nosocomial infections. The emergence of multiple-drug-resistantA. baumanniiisolates has increased in recent years. Directed toward phage therapy, a lytic phage ofA. baumannii, designated Abp53, was isolated from a sputum sample in this study. Abp53 has an isometric head and a contractile tail with tail fibers (belonging toMyoviridae), a latent period of about 10 min, and a burst size of approximately 150 PFU per infected cell. Abp53 could completely lyse 27% of theA. baumanniiisolates tested, which were all multiple drug resistant, but not other bacteria. Mg2+enhanced the adsorption and productivity of, and host lysis by, Abp53. Twenty Abp53 virion proteins were visualized in SDS-polyacrylamide gel electrophoresis, with a 47-kDa protein being the predicted major capsid protein. Abp53 has a double-stranded DNA genome of 95 kb. Sequence analyses of a 10-kb region revealed 8 open reading frames. Five of the encoded proteins, including 3 tail components and 2 hypothetical proteins, were similar to proteins encoded byA. baumanniistrain ACICU. ORF1176 (one of the tail components, 1,176 amino acids [aa]), which is also similar to tail protein gp21 ofKlebsiellaphage phiKO2, contained repeated domains similar to those within the ACICU_02717 protein ofA. baumanniiACICU and gp21. These findings suggest a common ancestry and horizontal gene transfer during evolution. As phages can expand the host range by domain duplication in tail fiber proteins, repeated domains in ORF1176 might have a similar significance in Abp53.

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Correlation of Checkerboard Synergy Testing with Time-Kill Analysis and Clinical Outcomes of Extensively Drug-Resistant Acinetobacter baumannii Respiratory Infections

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00981-16 ◽

2016 ◽

Vol 60 (11) ◽

pp. 6892-6895 ◽

Cited By ~ 12

Author(s):

Derek N. Bremmer ◽

Karri A. Bauer ◽

Stephanie M. Pouch ◽

Keelie Thomas ◽

Debra Smith ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Clinical Outcomes ◽

Clinical Utility ◽

Respiratory Infections ◽

Drug Resistant ◽

Content Type ◽

Extensively Drug Resistant ◽

Time Kill ◽

Synergy Testing

ABSTRACTWe tested 76 extensively drug-resistant (XDR)Acinetobacter baumanniiisolates by the checkerboard method using only wells containing serum-achievable concentrations (SACs) of drugs. Checkerboard results were correlated by time-kill assay and clinical outcomes. Minocycline-colistin was the best combinationin vitro, as it inhibited growth in one or more SAC wells in all isolates. Patients who received a combination that inhibited growth in one or more SAC wells demonstrated better microbiological clearance than those who did not (88% versus 30%;P= 0.025). The checkerboard platform may have clinical utility for XDRA. baumanniiinfections.

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Inhibition of LpxC Protects Mice from Resistant Acinetobacter baumannii by Modulating Inflammation and Enhancing Phagocytosis

mBio ◽

10.1128/mbio.00312-12 ◽

2012 ◽

Vol 3 (5) ◽

Cited By ~ 68

Author(s):

Lin Lin ◽

Brandon Tan ◽

Paul Pantapalangkoor ◽

Tiffany Ho ◽

Beverlie Baquir ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Gram Negative ◽

Content Type ◽

Novel Treatments ◽

New Class ◽

Virulent Strains ◽

Lethal Infection ◽

Tlr4 Activation ◽

Opsonophagocytic Killing

ABSTRACT New treatments are needed for extensively drug-resistant (XDR) Gram-negative bacilli (GNB), such as Acinetobacter baumannii. Toll-like receptor 4 (TLR4) was previously reported to enhance bacterial clearance of GNB, including A. baumannii. However, here we have shown that 100% of wild-type mice versus 0% of TLR4-deficient mice died of septic shock due to A. baumannii infection, despite having similar tissue bacterial burdens. The strain lipopolysaccharide (LPS) content and TLR4 activation by extracted LPS did not correlate with in vivo virulence, nor did colistin resistance due to LPS phosphoethanolamine modification. However, more-virulent strains shed more LPS during growth than less-virulent strains, resulting in enhanced TLR4 activation. Due to the role of LPS in A. baumannii virulence, an LpxC inhibitor (which affects lipid A biosynthesis) antibiotic was tested. The LpxC inhibitor did not inhibit growth of the bacterium (MIC > 512 µg/ml) but suppressed A. baumannii LPS-mediated activation of TLR4. Treatment of infected mice with the LpxC inhibitor enhanced clearance of the bacteria by enhancing opsonophagocytic killing, reduced serum LPS concentrations and inflammation, and completely protected the mice from lethal infection. These results identify a previously unappreciated potential for the new class of LpxC inhibitor antibiotics to treat XDR A. baumannii infections. Furthermore, they have far-reaching implications for pathogenesis and treatment of infections caused by GNB and for the discovery of novel antibiotics not detected by standard in vitro screens. IMPORTANCE Novel treatments are needed for infections caused by Acinetobacter baumannii, a Gram-negative bacterium that is extremely antibiotic resistant. The current study was undertaken to understand the immunopathogenesis of these infections, as a basis for defining novel treatments. The primary strain characteristic that differentiated virulent from less-virulent strains was shedding of Gram-negative lipopolysaccharide (LPS) during growth. A novel class of antibiotics, called LpxC inhibitors, block LPS synthesis, but these drugs do not demonstrate the ability to kill A. baumannii in vitro. We found that an LpxC inhibitor blocked the ability of bacteria to activate the sepsis cascade, enhanced opsonophagocytic killing of the bacteria, and protected mice from lethal infection. Thus, an entire new class of antibiotics which is already in development has heretofore-unrecognized potential to treat A. baumannii infections. Furthermore, standard antibiotic screens based on in vitro killing failed to detect this treatment potential of LpxC inhibitors for A. baumannii infections.

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Complete Genome Sequence of Acinetobacter radioresistens Strain LH6, a Multidrug-Resistant Bacteriophage-Propagating Strain

Microbiology Resource Announcements ◽

10.1128/mra.00929-18 ◽

2018 ◽

Vol 7 (5) ◽

Cited By ~ 2

Author(s):

Clay S. Crippen ◽

Steven Huynh ◽

William G. Miller ◽

Craig T. Parker ◽

Christine M. Szymanski

Keyword(s):

Antimicrobial Resistance ◽

Acinetobacter Baumannii ◽

Genome Sequence ◽

Complete Genome Sequence ◽

Related Species ◽

Complete Genome ◽

Multidrug Resistant ◽

Drug Resistant ◽

Content Type ◽

Resistance Determinants

Antimicrobial resistance is a major problem worldwide. Understanding the interplay between drug-resistant pathogens, such as Acinetobacter baumannii and related species, potentially acting as environmental reservoirs is critical for preventing the spread of resistance determinants.

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Cell Wall Changes in Amphotericin B-Resistant Strains from Candida tropicalis and Relationship with the Immune Responses Elicited by the Host

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02681-15 ◽

2016 ◽

Vol 60 (4) ◽

pp. 2326-2335 ◽

Cited By ~ 29

Author(s):

Ana C. Mesa-Arango ◽

Cristina Rueda ◽

Elvira Román ◽

Jessica Quintin ◽

María C. Terrón ◽

...

Keyword(s):

Immune Response ◽

Cell Wall ◽

Amphotericin B ◽

Candida Tropicalis ◽

Galleria Mellonella ◽

Regular Growth ◽

Cell Integrity ◽

Proinflammatory Response ◽

Content Type ◽

Resistant Strains

ABSTRACTWe have morphologically characterizedCandida tropicalisisolates resistant to amphotericin B (AmB). These isolates present an enlarged cell wall compared to isolates of regular susceptibility. This correlated with higher levels of β-1,3-glucan in the cell wall but not with detectable changes in chitin content. In line with this, AmB-resistant strains showed reduced susceptibility to Congo red. Moreover, mitogen-activated protein kinases (MAPKs) involved in cell integrity were already activated during regular growth in these strains. Finally, we investigated the response elicited by human blood cells and found that AmB-resistant strains induced a stronger proinflammatory response than susceptible strains. In agreement, AmB-resistant strains also induced stronger melanization ofGalleria mellonellalarvae, indicating that the effect of alterations of the cell wall on the immune response is conserved in different types of hosts. Our results suggest that resistance to AmB is associated with pleiotropic mechanisms that might have important consequences, not only for the efficacy of the treatment but also for the immune response elicited by the host.

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Synthetic Lethality Reveals Mechanisms of Mycobacterium tuberculosis Resistance to β-Lactams

mBio ◽

10.1128/mbio.01767-14 ◽

2014 ◽

Vol 5 (5) ◽

Cited By ~ 17

Author(s):

Shichun Lun ◽

David Miranda ◽

Andre Kubler ◽

Haidan Guo ◽

Mariama C. Maiga ◽

...

Keyword(s):

Cell Wall ◽

Mycobacterium Tuberculosis ◽

Synthetic Lethality ◽

Multidrug Resistant ◽

Cell Wall Biosynthesis ◽

Drug Resistant ◽

Content Type ◽

Innate Resistance ◽

Extensively Drug Resistant

ABSTRACT Most β-lactam antibiotics are ineffective against Mycobacterium tuberculosis due to the microbe’s innate resistance. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains has prompted interest to repurpose this class of drugs. To identify the genetic determinants of innate β-lactam resistance, we carried out a synthetic lethality screen on a transposon mutant library for susceptibility to imipenem, a carbapenem β-lactam antibiotic. Mutations in 74 unique genes demonstrated synthetic lethality. The majority of mutations were in genes associated with cell wall biosynthesis. A second quantitative real-time PCR (qPCR)-based synthetic lethality screen of randomly selected mutants confirmed the role of cell wall biosynthesis in β-lactam resistance. The global transcriptional response of the bacterium to β-lactams was investigated, and changes in levels of expression of cell wall biosynthetic genes were identified. Finally, we validated these screens in vivo using the MT1616 transposon mutant, which lacks a functional acyl-transferase gene. Mice infected with the mutant responded to β-lactam treatment with a 100-fold decrease in bacillary lung burden over 4 weeks, while the numbers of organisms in the lungs of mice infected with wild-type bacilli proliferated. These findings reveal a road map of genes required for β-lactam resistance and validate synthetic lethality screening as a promising tool for repurposing existing classes of licensed, safe, well-characterized antimicrobials against tuberculosis. IMPORTANCE The global emergence of multidrug-resistant and extensively drug-resistant M. tuberculosis strains has threatened public health worldwide, yet the pipeline of new tuberculosis drugs under development remains limited. One strategy to cope with the urgent need for new antituberculosis agents is to repurpose existing, approved antibiotics. The carbapenem class of β-lactam antibiotics has been proposed as one such class of drugs. Our study identifies molecular determinants of innate resistance to β-lactam drugs in M. tuberculosis, and we demonstrate that functional loss of one of these genes enables successful treatment of M. tuberculosis with β-lactams in the mouse model.

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Whole-Genome-Sequence-Based Characterization of Extensively Drug-Resistant Acinetobacter baumannii Hospital Outbreak

mSphere ◽

10.1128/msphere.00934-19 ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 3

Author(s):

Ghiwa Makke ◽

Ibrahim Bitar ◽

Tamara Salloum ◽

Balig Panossian ◽

Sahar Alousi ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Tertiary Hospital ◽

Outbreak Detection ◽

Whole Genome ◽

Transmission Network ◽

Drug Resistant ◽

Content Type ◽

Carbapenem Resistant ◽

Extensively Drug Resistant ◽

Long Read

ABSTRACT Carbapenem-resistant Acinetobacter baumannii (CRAB) is an important opportunistic pathogen linked to a variety of nosocomial infections and hospital outbreaks worldwide. This study aimed at investigating and characterizing a CRAB outbreak at a large tertiary hospital in Lebanon. A total of 41 isolates were collected and analyzed using pulsed-field gel electrophoresis (PFGE). Whole-genome sequencing (WGS) was performed on all the isolates, and long-read PacBio sequencing was used to generate reference genomes. The multilocus sequence types (MLST), repertoire of resistance genes, and virulence factors were determined from the sequencing data. The plasmid content was analyzed both in silico and using the A. baumannii PCR-based replicon typing (AB-PBRT) method. Genome analysis initially revealed two clones, one carrying blaOXA-23 on Tn2006 (ST-1305, ST-195, and ST-218) and another carrying blaOXA-72 on pMAL-1 (ST-502 and ST-2059, a new ST), with the latter having two subclones, as revealed using the Bayesian transmission network. All isolates were extensively drug resistant (XDR). WGS analysis revealed the transmission pathways and demonstrated the diversity of CRAB isolates and mobile genetic elements in this health care setting. Outbreak detection using WGS and immediate implementation of infection control measures contribute to restraining the spread and decreasing mortality. IMPORTANCE Carbapenem-resistant Acinetobacter baumannii (CRAB) has been implicated in hospital outbreaks worldwide. Here, we present a whole-genome-based investigation of an extensively drug-resistant CRAB outbreak rapidly spreading and causing high incidences of mortality at numerous wards of a large tertiary hospital in Lebanon. This is the first study of its kind in the region. Two circulating clones were identified using a combination of molecular typing approaches, short- and long-read sequencing and Bayesian transmission network analysis. One clone carried blaOXA-23 on Tn2006 (ST-1305, ST-195, and ST-218), and another carried blaOXA-72 on a pMAL-1 plasmid (ST-502 and ST-2059, a new ST). A pMAL-2 plasmid was circulating between the two clones. The approaches implemented in this study and the obtained findings facilitate the tracking of outbreak scenarios in Lebanon and the region at large.

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Determining the Development of Persisters in Extensively Drug-Resistant Acinetobacter baumannii upon Exposure to Polymyxin B-Based Antibiotic Combinations Using Flow Cytometry

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01712-19 ◽

2019 ◽

Vol 64 (3) ◽

Cited By ~ 4

Author(s):

Fiona Hui-Sian Wong ◽

Yiying Cai ◽

Hui Leck ◽

Tze-Peng Lim ◽

Jocelyn Qi-Min Teo ◽

...

Keyword(s):

Flow Cytometry ◽

Acinetobacter Baumannii ◽

Polymyxin B ◽

Live Cells ◽

Drug Resistant ◽

Bacterial Cells ◽

Persister Cells ◽

Content Type ◽

Initial Inoculum ◽

Extensively Drug Resistant

ABSTRACT Polymyxin B-based combinations are increasingly prescribed as a last-line option against extensively drug-resistant (XDR) Acinetobacter baumannii. It is unknown if such combinations can result in the development of nondividing persister cells in XDR A. baumannii. We investigated persister development upon exposure of XDR A. baumannii to polymyxin B-based antibiotic combinations using flow cytometry. Time-kill studies (TKSs) were conducted in three nonclonal XDR A. baumannii strains with 5 log10 CFU/ml bacteria against polymyxin B alone and polymyxin B-based two-drug combinations over 24 h. At different time points, samples were obtained and enumerated by viable plating and flow cytometry. Propidium iodide and carboxyfluorescein succinimidyl ester dyes were used to differentiate between live and dead cells and between dividing and nondividing cells, respectively, at the single-cell level, and nondividing live cells were resuscitated and characterized phenotypically. Our results from viable plating showed that polymyxin B plus meropenem and polymyxin B plus rifampin were each bactericidal (>99.9% kill compared to the initial inoculum) against 2/3 XDR A. baumannii strains at 24 h. By flow cytometry, however, none of the combinations were bactericidal against XDR A. baumannii at 24 h. Further analysis using cellular dyes in flow cytometry revealed that upon exposure to polymyxin B-based combinations, XDR A. baumannii entered a viable but nondividing persister state. These bacterial cells reinitiated division upon the removal of antibiotic pressure and did not have a growth deficit compared to the parent strain. We conclude that persister cells develop in XDR A. baumannii upon exposure to polymyxin B-based combinations and that nonplating methods appear to complement viable-plating methods in describing the killing activity of polymyxin B-based combinations against XDR A. baumannii.

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