The prevalence of antimicrobial resistance among Gram negative organisms is ever increasing. This rising abundance occurring simultaneously in animal and environmental populations coupled with the easy transmissibility of resistance mechanisms among species, and the paucity of novel Gram negative targeted agents in the antimicrobial pipeline, creates the consummate antimicrobial resistance threat. In this section, we will review the major mechanisms of resistance among Gram negative organisms with particular focus on clinically significant organisms causing healthcare associated infections. In 2013 report on antimicrobial resistance threats in the United States, the Centers for Disease Control or CDC identified resistant Gram negative organisms particularly Carbapenem-resistant Enterobacteriaceae as among the top three drug resistance threats to the United States. This report set the stage for increased focus on research and mitigation of resistant Gram negative organisms, especially in healthcare settings. It also highlighted the poor outcomes often associated with drug-resistant Gram negative infections, for example, approximately 50 percent of hospitalized patients with CRE bloodstream infections expired due to their infection. While this module is necessarily too short to cover every resistance mechanism and therapeutic option, we will review major trends in the evolution and epidemiology of Gram negative resistance, diagnosis, and therapy of resistant Gram negative organisms including key classes of Gram negative resistance, combination therapy, and recently approved therapeutic options. As was already alluded to in the introductory resistance module, the major mechanisms by which bacteria develop resistance include, one, selective pressure from antimicrobial use which allows for the selection of drug-resistant strains to emerge, and two, horizontal transfer of resistance including genetic material among bacteria, including from plasmids or other mobile genetic elements. Exchange of genetically mediated resistance mechanisms is extremely prevalent among Gram negative organisms and can result in a single organism simultaneously acquiring multiple resistance elements. Until recent years, much of the concern about Gram negative resistance has focused on expression of Extended-spectrum beta-lactamases abbreviated ESBLs. ESBLs are plasmid mediated enzymes that confer resistance to most beta-lactam based antibiotics, and they were first identified in the 1960s and increasingly recovered from patients especially in Europe throughout the 1980s and '90s. These resistant organisms were predominantly identified in hospitalized patients and a cause of great concern because of their ability to cause extensive outbreaks. Initial ESBL isolates arose as a consequence of mutations in the widespread plasmid encoded TEM-1 and SHV-1 beta-lactamases. To date, hundreds of different TEM and SHV varieties have been identified based on varying amino acids substitutions conferring resistance. Other varieties of ESBLs include, CTX-M and OXA in addition to other more rare or geographically restricted enzymes. ESBL producing Enterobacteriaceae had become widespread causes of HAIs, and are increasingly found as the cause of community acquired infections especially of the urinary tract. The widespread emergence of community associated ESBLs has been tied to the ascension of E-coli, a common member of the human gut flora as the predominant Enterobacteriaceae harboring the ESBL enzyme, and specifically to the emergence of CTX-M and ESBL carried by plasmids and expressed by environmental organisms that are easily acquired by E-coli. While global rates of community carriage of ESBL Enterobacteriaceae have exploded since 2008, carriage rates have varied geographically. Epidemiologists noting the global dissemination and distribution of ESBL producing organisms highlighted the significant intra and inter regional variations in ESBL carriage. Although, data acquisition and community surveillance differ markedly across the world, South-East Asia, the Western Pacific, and Eastern Mediterranean have all been identified as areas with a high prevalence of ESBL community carriage. Data from the African continent is limited and may not reflect the true burden of ESBL within this geographic region. Identifying areas with high rates of ESBL Enterobacteriaceae is important in assessing the risk for the spread of antimicrobial resistance by international travelers. In one study from the Netherlands, 34.3 percent of travelers who were initially ESBL negative acquired ESBL Enterobacteriaceae during international travel, particularly when they traveled to areas of high endemicity. ESBL producing Gram negative strains remain problematic and their emergence led to the increased use of carbapenems which are generally considered the antibiotics of choice for treating infections due to these organisms. Unfortunately, a newer problem has emerged namely, carbapenem-resistant Enterobacteriaceae abbreviated CRE, and other carbapenem-resistant Gram negatives. While multiple mechanisms may account for the carbapenem resistance phenotype, the main major concern since the early 1900s has been the emergence of enzymes that disable carbapenems. This mechanism was originally identified in pseudomonas and serratia in the early 1900s, and became widespread with the isolation of these enzymes from Klebsiella in the late 1900s. This discovery led to the well-recognized abbreviation KPC, which corresponds to Klebsiella pneumoniae producing carbapenemase. These enzymes efficiently hydrolyze nearly all beta-lactam antibiotics including carbapenems. Carbapenamases are no longer limited to Klebsiella pseudomonas and serratia and in fact, most Enterobacteriaceae can acquire this resistance mechanism through plasmid mediated acquisition of the enzyme. KPCs constitute one example of the Ambler class A enzymes. Many other carbapenamases belong to class B which includes the Metallo-beta-lactamases abbreviated, MBLs, while Amber Class D contains OXA type enzymes. These resistance enzymes have been identified in several different Gram negative genera including several of the Enterobacteriaceae family, Acinetobacter, and Pseudomonas. Among MBLs, NDM or New Delhi Metallo-beta-lactamases have garnered significant recent attention as a major emerging threat because of their easy transmissibility, thanks to a relatively stable plasmid backbone, and the association with medical tourism and other travel related exposure. Cephalosporin use remains a significant risk factor for acquisition and colonization with these resistant Gram negatives, and is thus a main focus of many stewardship efforts. These highly resistant organisms also present a unique threat to immunocompromised patients. A recent review of the global burden of carbapenem-resistant infections in neutropenic patients highlighted the emergence of carbapenem resistance among cancer patients over the last two decades, with the majority of the growth occurring since 2005. Further contributing to difficulty in the clinical management of carbapenemase producing bacterial isolates, is the fact that these organisms frequently also express resistance to other drug classes, for example, resistance to aminoglycosides and fluoroquinolones may be transmitted via the same plasmid. Alarmingly, this leads to drastically limited therapeutic options. Additional mechanisms to avert antimicrobial therapy employed by Gram-negative bacteria include porin mutation, efflux pumps, and inducible ampC beta-lactamase enzymes. For example, "SPICE" organisms chromosomally encode an inducible beta-lactamase enzyme called ampC. Members of this group include Serratia, Providencia, indole-positive Proteus species, Citrobacter, and Enterobacter, although both Acinetobacter and Pseudomonas may also carry genes that include ampC. AmpC, whose product is predominantly a cephalosporinase, and is not inhibited by most available beta-lactamase inhibitors, may also be carried and expressed in plasmids. The major importance of the inducible ampC is that, particularly in Enterobacter, small numbers of de-repressed mutants can constitutively produce the enzyme in large amounts. These mutants can be selected during treatment with third-generation cephalosporins. Concerns about treatment failure due to this mechanism account for the admonition against the use of third-generation cephalosporins in the treatment of serious Enterobacter infections. ESBLs may also be present in SPICE organisms, further complicating both laboratory identification and antimicrobial therapy. It can be difficult to phenotypically distinguish among these mechanisms depending on the method of laboratory identification employed. For example, it can be quite difficult to differentiate among resistance due to chromosomal ampC, drug impermeability, and carbapenemase production. The emergence of widespread resistance among Enterobacteriaceae, as well as other Gram-negative organisms prompted the Clinical and Laboratory Standards Institute, abbreviated CLSI, and its European counterpart, EUCAST, to reevaluate the breakpoint criteria for determining susceptibility of organisms to several antimicrobials. Although carbapenems remain the mainstay of therapy for ESBL-producing organisms, therapeutic options for carbapenemase-producing bacteria are less clear. Clinicians must choose from a limited array of remaining antibiotics, most likely to be active, and these choices often include drugs with significant toxicities and side effect profiles. For example, gentamicin, tigecycline, colistin, and polymyxin B. Combination therapy with multiple agents, often including paradoxically treatment with an optimally dosed carbapenem, appears to have some clinical advantage over monotherapy, particularly when it comes to engendering resistance, and may perhaps have a mortality benefit. The data supporting combination therapy is derived mostly from observational retrospective cohorts and outbreak investigations with all the limitations that those types of studies entail. The most common combination therapies include a carbapenem plus tigecycline and either an aminoglycosides or a polymyxin. Other agents have also been reported as part of combination regimens for treatment of carbapenem-resistant infections, including rifampin based on in vitro data and fosfomycin, the IV formulation of which is not available in the US. Aztreonam has also been included in combination regimens despite high-level resistance in order to exploit its competitive inhibition of MBLs. Ultimately, the selection of components of combination therapy likely depends on patient clinical characteristics such as site of infection and the type of carbapenemase being produced. Infections caused by multidrug-resistant Acinetobacter have extremely limited therapeutic options, often restricted to polymyxin, tigecyclin, and sulbactam. Limited studies of these agents used either as monotherapy or in combination, including synergistic combination therapy, have been reported and seemingly, there's no obvious first-line therapy. Finally, minocycline, initially available in the 1960s and voluntarily withdrawn from the US market in the IV formulation in 2005, reappeared as a therapeutic option for MDR infections in 2009. Various synergistic combinations of minocycline with a variety of antimicrobials have been evaluated in vitro and in vivo, but never in a randomized controlled trial. While many older antibiotics have been resurrected for the management of resistant Gram-negative organisms, two new agents have recently come to market for the treatment of intra-abdominal infections and complicated urinary tract infections. Because of limited therapeutic options for Gram-negative resistant organisms, they have been used in patients with highly resistant Gram-negative infection at other sites and who have failed other therapies. Ceftazidime-avibactam is a new beta-lactam plus inhibitor combination therapy employing a novel beta-lactamase agent. Limited anecdotal clinical data hints that it may hold promise for the treatment of infections due to carbapenemase producers, while laboratory data suggests this agent has inhibitory capability across multiple Ambler classes, including KPCs and possibly some MBLs. Ceftolozane-tazobactam is a novel cephalosporin plus beta-lactamase inhibitor combination, with enhanced stability against ampC enzymes, and typically exhibits a lower MIC to Pseudomonas species. Coverage of ampC-containing organisms is not uniform, however, and this agent does not have activity against two of the most worrisome mechanisms of Gram-negative resistance, KPCs and MBLs. Thus, limiting its therapeutic utility. In conclusion, antimicrobial resistance and Gram-negative organisms is a worrisome threat, especially in the context of healthcare-associated infections, due to the multiplicity of resistance mechanisms and their easy transmissibility among multiple species. The problem of Gram-negative resistance is further complicated by extremely limited novel therapeutic options and a substantial side effect profiles of resurrected antimicrobial therapies. Mitigation and management of Gram-negative resistance depends in large part on the prudent use of existing antibiotics, attention to appropriate laboratory diagnosis, the use of combination therapy, and standard infection control, and stewardship practices.
