Summary: AmpC β-lactamases are clinically important cephalosporinases encoded on the chromosomes of many of the Enterobacteriaceae and a few other organisms, where they mediate resistance to cephalothin, cefazolin, cefoxitin, most penicillins, and β-lactamase inhibitor-β-lactam combinations. In many bacteria, AmpC enzymes are inducible and can be expressed at high levels by mutation. Overexpression confers resistance to broad-spectrum cephalosporins including cefotaxime, ceftazidime, and ceftriaxone and is a problem especially in infections due to Enterobacter aerogenes and Enterobacter cloacae, where an isolate initially susceptible to these agents may become resistant upon therapy. Transmissible plasmids have acquired genes for AmpC enzymes, which consequently can now appear in bacteria lacking or poorly expressing a chromosomal blaAmpC gene, such as Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis. Resistance due to plasmid-mediated AmpC enzymes is less common than extended-spectrum β-lactamase production in most parts of the world but may be both harder to detect and broader in spectrum. AmpC enzymes encoded by both chromosomal and plasmid genes are also evolving to hydrolyze broad-spectrum cephalosporins more efficiently. Techniques to identify AmpC β-lactamase-producing isolates are available but are still evolving and are not yet optimized for the clinical laboratory, which probably now underestimates this resistance mechanism. Carbapenems can usually be used to treat infections due to AmpC-producing bacteria, but carbapenem resistance can arise in some organisms by mutations that reduce influx (outer membrane porin loss) or enhance efflux (efflux pump activation).

AmpC β-Lactamases

The global emergence of multidrug-resistant Gram-negative bacteria is a growing threat to antibiotic therapy. The chromosomally encoded drug efflux mechanisms that are ubiquitous in these bacteria greatly contribute to antibiotic resistance and present a major challenge for antibiotic development. Multidrug pumps, particularly those represented by the clinically relevant AcrAB-TolC and Mex pumps of the resistance-nodulation-division (RND) superfamily, not only mediate intrinsic and acquired multidrug resistance (MDR) but also are involved in other functions, including the bacterial stress response and pathogenicity. Additionally, efflux pumps interact synergistically with other resistance mechanisms (e.g., with the outer membrane permeability barrier) to increase resistance levels. Since the discovery of RND pumps in the early 1990s, remarkable scientific and technological advances have allowed for an in-depth understanding of the structural and biochemical basis, substrate profiles, molecular regulation, and inhibition of MDR pumps. However, the development of clinically useful efflux pump inhibitors and/or new antibiotics that can bypass pump effects continues to be a challenge. Plasmid-borne efflux pump genes (including those for RND pumps) have increasingly been identified. This article highlights the recent progress obtained for organisms of clinical significance, together with methodological considerations for the characterization of MDR pumps.

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The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

The pathogenesis of Legionella pneumophila is derived from its growth within lung macrophages after aerosols are inhaled from contaminated water sources. Interest in this bacterium stems from its ability to manipulate host cell vesicular-trafficking pathways and establish a membrane-bound replication vacuole, making it a model for intravacuolar pathogens. Establishment of the replication compartment requires a specialized translocation system that transports a large cadre of protein substrates across the vacuolar membrane. These substrates regulate vesicle traffic and survival pathways in the host cell. This Review focuses on the strategies that L. pneumophila uses to establish intracellular growth and evaluates why this microorganism has accumulated an unprecedented number of translocated substrates that are targeted at host cells.

/pdf/the-legionella-pneumophila-replication-vacuole-making-a-cosy-2kbr827dp5.pdf

The Legionella pneumophila replication vacuole: making a cosy niche inside host cells

Macrophages from C57BL/6J (B6) mice restrict growth of the intracellular bacterial pathogen Legionella pneumophila. Restriction of bacterial growth requires caspase-1 and the leucine-rich repeat-containing protein Naip5 (Birc1e). We identified mutants of L. pneumophila that evade macrophage innate immunity. All mutants were deficient in expression of flagellin, the primary flagellar subunit, and failed to induce caspase-1-mediated macrophage death. Interestingly, a previously isolated flagellar mutant (fliI) that expresses, but does not assemble, flagellin did not replicate in macrophages, and induced macrophage death. Thus, flagellin itself, not flagella or motility, is required to initiate macrophage innate immunity. Immunity to Legionella did not require MyD88, an essential adaptor for toll-like receptor 5 (TLR5) signaling. Moreover, flagellin of Legionella and Salmonella induced cytotoxicity when delivered to the macrophage cytosol using Escherichia coli as a heterologous host. It thus appears that macrophages sense cytosolic flagellin via a TLR5-independent pathway that leads to rapid caspase-1-dependent cell death and provides defense against intracellular bacterial pathogens.

/pdf/flagellin-deficient-legionella-mutants-evade-caspase-1-and-3exfwu2amr.pdf

Flagellin-deficient Legionella mutants evade caspase-1- and Naip5-mediated macrophage immunity.

Francisella tularensis is a highly infectious aerosolizable intracellular pathogen that is capable of causing a debilitating or fatal disease with doses as low as 25 colony-forming units. There is no licensed vaccine available. Since the 1950s there has been concern that F. tularensis could be used as a biological threat agent, and it has received renewed attention recently owing to concerns about bioterrorism. The International Conference on Tularaemia in 2003 attracted more than 200 delegates, twice the number of participants as previous meetings. This is a reflection of the increased funding of research on this pathogen, particularly in the United States.

Tularaemia: bioterrorism defence renews interest in Francisella tularensis

We present the genomic sequence of Legionella pneumophila, the bacterial agent of Legionnaires' disease, a potentially fatal pneumonia acquired from aerosolized contaminated fresh water The genome includes a 45-kilobase pair element that can exist in chromosomal and episomal forms, selective expansions of important gene families, genes for unexpected metabolic pathways, and previously unknown candidate virulence determinants We highlight the genes that may account for Legionella's ability to survive in protozoa, mammalian macrophages, and inhospitable environmental niches and that may define new therapeutic targets

http://science.sciencemag.org/content/sci/305/5692/1966.full.pdf

The genomic sequence of the accidental pathogen Legionella pneumophila.

This chapter aims at obtaining the complete sequence of the Philadelphia 1 strain of Legionella pneumophila, derived from the original 1976 outbreak that established the clinical entity known as Legionnaires' disease. The GC content of L. pneumophila is ~38% over most of its length. From the genomic sequence so far (more than 85% of the sequence is currently contained in contigs greater than 1 kb), 3,000 open reading frames (ORF) were covered, about 2,000 of which are complete, and on the basis of homology to genes from other prokaryotes, more than 1,100 putative genes in L. pneumophila were identified. Genes in L. pneumophila Philadelphia 1 can be compared with genes in other pathogenic organisms. The amino acid sequence can guide structural and functional analyses of enzymes and other proteins of the organism. The authors used knockout strategies in the past to assess the function of genes discovered in the course of the sequencing project; and this continues to be their preferred method for functional analysis.

The Legionella pneumophila Sequencing Project

Charcot-Marie-Tooth disease (CMT) is a genetically and clinically heterogeneous group of inherited neuropathies. Monoallelic pathogenic variants in ATP1A1 were associated with axonal and intermediate CMT. ATP1A1 encodes for the catalytic α1 subunit of the Na+/ K+ ATPase. Besides neuropathy, other associated phenotypes are spastic paraplegia, intellectual disability, and renal hypomagnesemia. We hereby report the first demyelinating CMT case due to a novel ATP1A1 variant.Whole-exome sequencing on the patient's genomic DNA and Sanger sequencing to validate and confirm the segregation of the identified p.P600R ATP1A1 variation were performed. To evaluate functional effects, blood-derived mRNA and protein levels of ATP1A1 and the auxiliary β1 subunit encoded by ATP1B1 were investigated. The ouabain-survival assay was performed in transfected HEK cells to assess cell viability, and two-electrode voltage clamp studies were performed in Xenopus oocytes.The variant was absent in the local and global control datasets, falls within a highly conserved protein position, and is in a missense-constrained region. The expression levels of ATP1A1 and ATP1B1 were significantly reduced in the patient compared to healthy controls. Electrophysiology indicated that ATP1A1p.P600R injected Xenopus oocytes have reduced Na+/ K+ ATPase function. Moreover, HEK cells transfected with a construct encoding ATP1A1p.P600R harbouring variants that confers ouabain insensitivity displayed a significant decrease in cell viability after ouabain treatment compared to the wild type, further supporting the pathogenicity of this variant.Our results further confirm the causative role of ATP1A1 in peripheral neuropathy and broaden the mutational and phenotypic spectrum of ATP1A1-associated CMT. 

/pdf/the-phenotypic-spectrum-of-pathogenic-atp1a1-variants-286yx4wt.pdf

Anthi Georghiou

Papers

The genomic sequence of the accidental pathogen Legionella pneumophila.

The Legionella pneumophila Sequencing Project

The phenotypic spectrum of pathogenic ATP1A1 variants expands: the novel p.P600R substitution causes demyelinating Charcot–Marie–Tooth disease