Robert A. Bonomo

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

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. 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.

First Report of a Verona Integron-Encoded Metallo-β-Lactamase-Producing Klebsiella pneumoniae Infection in a Child in the United States

We report the first case of a child in the United States infected with an organism producing a Verona Integron-Encoded Metallo-β-Lactamase. This child succumbed to a ventilator-associated pneumonia caused by a Klebsiella pneumoniae producing this resistance mechanism.

Acinetobacter baumannii and Acinetobacter spp.

Acinetobacter baumannii is a bacterial pathogen increasingly identified in the clinical microbiology laboratory as a cause of infection in humans. Upon microscopic examination, A. baumannii appears as a Gram-negative coccobacillus, and it produces clear colonies when grown on MacConkey agar, indicating its inability to ferment lactose. The taxonomy of Acinetobacter genus, part of the γ subclass of the Proteobacteria phylum, is complex and currently comprises 43 species (http://www.bacterio.net/acinetobacter.html), defined mostly by genomic DNA–DNA hybridization.

Chapter 12 – Non-Phenotypic Tests to Detect and Characterize Antibiotic Resistance Mechanisms in Enterobacteriaceae

In the past two decades, we have observed a rapid emergence of multidrug-resistant Enterobacteriaceae. These isolates possess genes encoding for extended-spectrum β-lactamases (ESBLs) or plasmid-mediated AmpCs that confer resistance to last-generation cephalosporins. Furthermore, other resistance traits against quinolones and aminoglycosides are also frequently co-associated. Even more concerning is the rapid increase of Enterobacteriaceae carrying genes conferring resistance to carbapenems. Unfortunately, standard microbiological procedures require several days to isolate the responsible pathogen and to provide correct antimicrobial susceptibility test results. Thus, there is emerging interest in the early and more sensitive detection of resistance mechanisms. In this chapter, we present a summary of the most advanced methods (e.g., multiplex PCRs, real-time PCRs, microarrays, MALDI-TOF MS, and PCR/ESI MS) presently available for the rapid detection of resistance genes in Enterobacteriaceae. Taking into account speed, manageability, accuracy, versatility, and costs, the possible settings of application of these methods and their superiority against standard phenotypic methods are discussed.