MDRO Infections: Gram-Negative Organisms

John P. Mills

Owen R. Albin

Laraine Washer

Lona Mody

Keith S. Kaye

MECHANISMS AND EPIDEMIOLOGY OF GRAM-NEGATIVE BACTERIAL RESISTANCE

General Principles

Major mechanisms of antibiotic resistance among Gramnegative bacteria (GNB) include enzymatic inactivation of antibiotics, decreased bacterial membrane permeability, upregulation of drug efflux pumps, and target site alterations. While antimicrobial resistance may occur as the result of de novo mutations, the acquisition of resistance mechanisms among contemporary GNB is increasingly mediated by the transfer of mobile genetic elements (ie, transposons, integrons, plasmids, and bacteriophages) within and between bacterial populations. While specific mechanisms of resistance predominate among particular GNB, many pathogens exhibit multiple resistance mechanisms simultaneously, some of which may evolve during antimicrobial treatment. This section highlights major mechanisms of resistance encountered in multidrug-resistant Gram-negative bacteria (MDR-GNB), with special attention to beta-lactamases (Table 20-1). For a more comprehensive review of GNB antimicrobial resistance mechanisms, readers are referred elsewhere.¹ In addition, the clinical epidemiology of certain MDR-GNB strains of importance in hospitals and as a cause of healthcare-associated infections (HAIs) is reviewed.

Enterobacteriaceae

Beta-Lactamases The major determinants of antimicrobial resistance among Enterobacteriaceae are beta-lactamases, a set of predominantly plasmid-encoded enzymes capable of hydrolyzing the beta-lactam ring of various penicillins, cephalosporins, monobactams, and carbapenems. These enzymes exhibit tremendous diversity with respect to hydrolytic spectrum of activity, susceptibility to betalactamase inhibitors and plasmid or chromosomal localizations.² Contemporary categorization of beta-lactamases utilizes the Ambler classification scheme, which groups enzymes based on amino acid homology rather than phenotypic characteristics.

Extended-Spectrum Beta-Lactamases Early beta-lactam resistance among Enterobacteriaceae was mediated by plasmid-mediated enzymes with activity against early-generation, narrow-spectrum penicillins and cephalosporins. By the 1970s, these beta-lactamases (ie, TEM-1 and, to a lesser extent, TEM-2 and SHV-1 types) had disseminated among Enterobacteriaceae, Haemophilus influenzae, and Neisseria gonorrhoeae, necessitating the development of antimicrobial agents capable of withstanding degradation from these enzymes. These “extended-spectrum” agents, particularly, the novel cephalosporins coupled with an oxymino-side chain (ie, cefotaxime, ceftazidime, ceftriaxone, cefepime) gained widespread clinical use beginning in the 1980s, in turn exerting novel selection pressure on microbial pathogens and precipitating the emergence of extended-spectrum beta-lactamases (ESBLs) in the 1980s.³

The earliest described ESBLs among Enterobacteriaceae were TEM-derived and SHV-derived types, which occured via amino acid substitutions in their precursor namesake enzymes, occurring in the 1980s. More prevalent among contemporary Enterobacteriaceae are the CTX-M family of ESBLs, named for their hydrolytic efficacy against cefotaxime. Unlike TEM- and SHV-derived types, CTX-M ESBLs likely emerged via plasmid acquisition from environmental bacteria. They have notably greater hydrolytic activity against cefepime than do comparator ESBLs and are more potently inhibited by tazobactam than clavulanic acid.⁴ CTX-M-producing Enterobacteriaceae have become the leading causes of nosocomial and community-acquired ESBL infections globally; the ST131 Escherichia coli clone in particular, which carries a CTX-M-15 beta-lactamase in concert with fluoroquinolone resistance determinants, is responsible for the majority of multidrug-resistant E coli infections worldwide.⁵^,⁶

Carbapenemases Carbapenemases, as their name suggests, are beta-lactamases with the ability to hydrolyze carbapenems, in addition to penicillins, cephalosporins, and (for the most part) monobactams. In addition to hydrolyzing carbapenems, most carbapenemases effectively neutralize precursor beta-lactams and resist inhibition of older-generation beta lactamase inhibitors (BLIs). Metallo-carbapenemases (Ambler Class B enzymes) utilize zinc at their active site to facilitate hydrolysis and serine-carbapenemases (Ambler Class A or, rarely, Ambler Class D enzymes) utilize a serine residue at their active site for the same purpose.
While chromosomally encoded carbapenemases capable of clonal spread were recognized sporadically prior to the 1990s, carbapenem resistance among Enterobacteriaceae reached epidemic proportions with the emergence of plasmid-mediated carbapenemases capable of global propagation and interspecies dispersion (Table 20-2).

TABLE 20-1 Multidrug-Resistant Gram-Negative Bacteria (MDR-GNB) Abbreviations

	*Abbreviation*	*Definition*	*Examples*
Multidrug-resistant Gram-negative bacteria
	MDR-GNB	Gram-negative bacteria phenotypically resistant to ≥3 classes of antimicrobials
Enterobacteriaceae
Carbapenem-resistant Enterobacteriaceae	CRE	Enterobacteriaceae phenotypically resistant to ≥ 1 carbapenem OR detection of carbapenemase
Nonfermenting Gram-negative bacteria
	NF-GNB	Gram-negative bacteria incapable of fermenting glucose	P aeruginosa, A baumannii, S maltophilia, Burkholderia cepacia complex
Carbapenem-resistant Pseudomonas aeruginosa	CRPA	P aeruginosa phenotypically resistant to ≥ 1 carbapenem
Carbapenem-resistant Acinetobacter baumannii	CRAB	A baumannii phenotypically resistant to ≥ 1 carbapenem
Beta-lactamases
Extended spectrum beta-lactamases	ESBLs	Enzymes capable of hydrolyzing 3rd and fourth-generation cephalosporins in addition to penicillin agents	CTX-M, TEM, SHV
Carbapenemase-producing organisms	CPOs	Gram-negative bacteria that produce enzymes capable of hydrolyzing carbapenems	KPC, NDM, OXA (see Table 20-2)

TABLE 20-2 Epidemiology of Carbapenemase Enzymes

*Enzyme*	*Genetic platform*	*Species*	*Geographic distribution*	*Misc*
KPC (Klebsiella pneumonia carbapenemase)	Plasmid	Enterobacteriaceae (K pneumoniae, E coli, Enterobacter, etc.) >Pseudomonas	United States, Greece, Israel, Italy, China, South America	Most frequent cause of carbapenem resistance in the United States
NDM (New Delhi metallo-beta-lactamase)	Plasmid	Enterobacteriaceae >>Pseudomonas, Acinetobacter	Indian subcontinent and Balkan region	Occasionally retain activity to aztreonam
OXA-48 (oxacillinase)	Plasmid	Enterobacteriaceae	Southern and Western Europe, Turkey, North Africa	Difficult to detect phenotypically
VIM (Verona integron-encoded MBL)	Plasmid	Enterobacteriaceae >Pseudomonas	Italy, Greece, East Asia	Metallo-beta-lactamase
IMP	Plasmid	Enterobacteriaceae >Pseudomonas	East Asia and South America	Metallo-beta-lactamase
SME	Chromosome	Serratia marcescens	Sporadic in North and South America

Ambler Class A Carbapenemases The first serine carbapenemases were observed among Enterobacteriaceae as early as 1982 from isolates of Serratia marcescens (harboring SME-1 carbapenemases) and Enterobacter cloacae (harboring IMI and NMC-A carbapenemases). These enzymes were chromosomally encoded and were infrequently implicated in outbreak settings. The emergence in 1996 of a plasmid-encoded serine carbapenemase from a Klebsiella pneumoniae isolate capable of epidemic dispersion and interspecies transfer has played a larger role in the current crisis in carbapenem resistance
among Enterobacteriaceae. These Klebsiella pneumoniae carbapenemases (KPCs) have subtle modifications in their hydrolytic active site in comparison to SME-1, IMI, and NMC-A carbapenemases, markedly altering their substrate specificity. KPC carbapenemases neutralize beta-lactams of all classes: they demonstrate efficient hydrolysis of penicillins and early-generation cephalosporins, in addition to attenuated but clinically significant hydrolysis of imipenem, meropenem, aztreonam, and extended-spectrum cephalosporins. They remain only marginally inhibited by traditional BLIs (clavulanate, sulbactam, and tazobactam). KPC and its enzymatic variants reside on transferable plasmids, a feature that has facilitated their global proliferation and interspecies dispersion. Indeed, these carbapenemases have become endemic in some healthcare settings and precipitated numerous country-wide outbreaks globally. KPC-family carabapenemases have since been detected in other Enterobacteriaceae including E coli and Enterobacter spp. as well as other GNB.⁷

Ambler Class B Carbapenemases Metallo-beta-lactamases (MBLs) among Enterobacteriaceae are characterized by complete resistance to all commercially available BLIs (including avibactam, vaborbactam, and relebactam) and inhibition by metal ion chelators (including EDTA). They have a broad substrate spectrum, hydrolyzing all known beta-lactams with the notable exception of aztreonam. MBL-producing organisms, however, often coexpress other resistance factors (ESBLs or ampC-beta-lactamases), rendering aztreonam ineffective. Similar to their serine-beta-lactamase counterparts, genetically transferable families of MBLs (IMP- and VIM-types) have rapidly emerged and spread among Enterobacteriaceae since the 1990s, particularly among K pneumoniae. In 2009, a novel MBL, the New Delhi metallo-beta-lactamase-1 (NDM-1), was isolated from a Swedish patient hospitalized in India. Sporadic cases of infections due to NDM-producing Enterobacteriaceae were subsequently noted in patients with healthcare exposure in India.

Ambler Class D Carbapenemases OXA-type beta-lactamases are a group of homologous enzymes, so named for their ability to hydrolyze oxacillin, that figure prominently in beta-lactam resistance among Pseudomonas and Acinetobacter spp. One OXA-type enzyme in particular, OXA-48, has become widespread among K. pneumoniae and other Enterobacteriaceae. While OXA-48 exhibits relatively modest carbapenem hydrolysis on its own, it generates markedly elevated carbapenem minimum inhibitory concentrations (MICs) in vitro when cloned into previously susceptible strains of E coli lacking OmpF and OmpC porin channels, indicating synergistic contributions to carbapenem resistance among Gram-negative isolates carrying multiple resistance determinants.⁸

AmpC Beta-Lactamases AmpC beta-lactamases are Ambler Class C enzymes capable of hydrolyzing penicillins, cephalosporins, and monobactams. The majority of clinically relevant AmpC beta-lactamases are chromosomally encoded in a number of Enterobacteriaceae, most notably E cloacae, Enterobacter aerogenes, Citrobacter freundii, S marcescens, Providencia stuartii, and Morganella morganii. Unlike ESBLs, AmpC enzymes are not inhibited by traditional BLIs and are sometimes produced in clinically relevant concentrations in response to beta-lactam exposure. Many organisms that chromosomally encode AmpC beta-lactamases may initially appear susceptible to β-lactam agents, but after exposure to certain agents (such as third-generation cephalosporins) they can rapidly develop broad-spectrum β-lactam resistance.⁹ This phenomenon occurs most commonly with E cloacae followed by C freundii and S marcescens.

The molecular pathways regulating AmpC production are complex but generally involve inhibition of the AmpR transcriptional repressor, either in response to beta-lactam cell wall degradation products or as a product of mutations in the AmpR regulatory apparatus in clonal subpopulations. Potent inducers of AmpC production include aminopenicillins, amoxicillin-clavulanate, narrow-spectrum cephalosporins, and cephamycins, whereas weak inducers include piperacillin-tazobactam, aztreonam, and extendedspectrum cephalosporins. Cefepime is a weak AmpC inducer, and carbapenems are universally poor substrates for AmpC hydrolysis.

Non-Beta-Lactamase-Mediated Resistance The emergence of quinolone resistance among Enterobacteriaceae over the last two decades has been particularly staggering. According to data from the National Healthcare Safety Network (NHSN), over 40% of E coli central line-associated bloodstream infection (CLABSI) isolates in 2010 were quinolone resistant. Quinolone resistance among Enterobacteriaceae is frequently due to chromosomal point mutations in quinolone resistance-determining regions (QRDRs) of the quinolone target enzymes, DNA gyrase and topoisomerase IV, conferring altered structural confirmations that reduce drug binding to the enzyme-DNA complex. This is the predominant mechanism of resistance observed in the globally disseminated quinolone-resistant ESBL E coli clone, ST131 H30. Additionally, transferrable mechanisms of quinolone resistance among Enterobacteriaceae have emerged over the last two decades. Plasmids carrying the qnrA gene, expression of which prevents fluoroquinolone binding to DNA gyrase, have been detected in as many as 11% of K pneumonia strains in the United States.¹⁰

Aminoglycosides have remained relatively effective against Enterobacteriaceae; however, resistance has been increasingly detected, particularly among ESBL-producing and carbapenem-resistant Enterobacteriaceae (CRE) strains, which often carry aminoglycoside-modifying enzymes in concert with beta-lactamases. Resistance determinants against trimethoprim-sulfamethoxazole (TMP-SMX) are increasingly observed in multidrug-resistant coliforms as well, often due to transferrable variants of the dihydrofolate reductase (dhfr) gene expressed in conjunction with beta-lactamases.

MDR Enterobacteriaceae Clinical Epidemiology The world pandemic of CTX-M-producing E coli has resulted in a new epidemiology for MDR Enterobacteriaceae. Opportunistic healthcare-associated outbreaks with mainly single clones of SHV- and TEM-type ESBL-producing K pneumoniae have been replaced by sporadic and epidemic community infections with clones of more virulent MDR CTX-M-producing E coli. Spread has typically occurred among healthy older adults at home and in long-term care
facilities; admission of these types of individuals to hospital or care homes may result in healthcare-associated outbreaks. In recent years, community-onset infections due to ESBL-producing ST131 E coli have become common.¹¹ Of note, CTX-M is also commonly isolated among ESBL-producing K pneumonia.

CRE epidemiology is complex as the prevalence of particular carbapenemase genes varies substantially between different geographic locations and changes over time. Since initial detection of KPC along the eastern seaboard of the United States, CRE have since established endemicity in much of the world, with particularly high rates found in Israel, Greece, Italy, China, and much of South America. NDM has also disseminated rapidly, originating in South Asia and then spreading worldwide, with endemicity in South and East Asia as well as the Middle East. While initially only associated with healthcare exposure, new NDM community-acquired cases are occurring with increasing frequency in South Asia. A prior study of water sources in India found NDM-producing Enterobacteriaceae in a large percentage of seepage water sites, suggesting large-scale environmental contamination with the potential to cause community-acquired infections.¹² OXA-48 is widespread in much of Europe, North Africa, and the Middle East. There have been increasing reports in recent years of Enterobacteriaceae coproducing OXA-48 and other carbapenemases, typically NDM or KPC. KPC remains the most common type of carbapenemase in the United States, but NDM and OXA-48-producing strains of Enterobacteriaceae are also present in the United States.

Patients at highest risk for CRE infections are those with impaired functional status, multiple comorbidities, prior hospitalization or long-term care stay, prior antibiotic exposure, and indwelling devices.¹³ Long-term acute care hospitals (LTACHs), which provide care for patients with intensive medical needs, such as those requiring chronic ventilatory support, have been identified as a reservoir for CRE and can serve as a local amplifier of CRE transmission. CRE can spread rapidly within a facility as well as regionally, among acute care hospitals and LTACs.¹⁴ Infections due to CRE are associated with poor outcomes due to a variety of reasons, including comorbid conditions, delays in time to appropriate antimicrobial treatment, and the necessity of use of second and third-line antibiotic treatment options, often with notable toxicity. A meta-analysis reported a twofold increase in mortality among patients infected with CRE vs carbapenem-susceptible Enterobacteriaceae.¹⁵ Attributable mortality to CRE was highly variable, ranging from 3%-44%.

Polymyxin resistance was historically due to sporadic chromosomal mutations leading to alterations of the lipid A component of lipopolysaccharide (LPS). In 2016, the first cases of mcr-1, an LPS-modifying enzyme located on a transmissible plasmid, were reported among human and poultry/swine E coli isolates in China.¹⁶ Since then, infections due to mcr-1 Enterobacteriaceae have been identified worldwide. There have subsequently been numerous reports of infections due to Enterobacteriaceae coproducing carbapenemases (predominantly NDM and OXA-48) and mcr-1.¹⁷

Nonfermenting Gram-Negative Bacilli

Antimicrobial resistance mechanisms among major nosocomial nonfermenting Gram-negative bacteria (NF-GNB) (Pseudomonas aeruginosa, Acinetobacter baumannii group, Stenotrophomonas maltophilia, and Burkholderia spp.) differ from those observed in Enterobacteriaceae in a number of respects. First nonhydrolytic mechanisms of resistance (eg, porin channel mutations and drug efflux pumps) play a proportionally larger role. Second, the bacterial outer membrane among NF-GNB is less permeable to hydrophilic antibiotics relative to that of Enterobacteriaceae. Finally, NF-GNB possess a unique ability to form biofilms on conducive environmental surfaces and, in particular, populations (eg, patients with impaired immunity, structurally abnormal lung architecture, and/or indwelling prosthetic devices). Biofilms impede antibiotic penetration, modify bacterial growth kinetics, and generate the presence of nonreplicating persister cells.

Pseudomonas aeruginosa

Owing to its inherent defense mechanisms, genetic versatility, and ability to coregulate multiple resistance factors, P aeruginosa remains at the forefront of antimicrobial resistance. Resistance among P aeruginosa is rising in HAIs: according to the NHSN, MDR isolates (defined as an isolate that is nonsusceptible to at least one agent in three or more antibiotic classes) were identified in nearly 20% of cases of ventilator-associated pneumonia (VAP), CLABSIs, and catheter-associated urinary tract infections (CAUTIs) caused by P aeruginosa from 2011 to 2014.¹⁸ While susceptibility profiles of Pseudomonas spp. vary across geographic regions, rates of quinolone and beta-lactam resistance have been noted to exceed 40% in specific locales.¹⁹

Inherent Resistance Inherent defenses among wild-type P aeruginosa strains include basal production of AmpC beta-lactamases and membrane impermeability to antibiotics. The outer membrane of Pseudomonas is 92% less permeable to hydrophilic compounds than that of E coli, likely in part due to the inherent biochemical composition of its membrane phospholipid structure and less permissive porin membrane channels. Porin channels in isolates of P aeruginosa are generally smaller and more substrate-specific than those found in E coli. Unsurprisingly, wild-type Pseudomonas isolates exhibit higher baseline MICs to quinolones and beta-lactams than Enterobacteriaceae.

Chromosomally Mediated Resistance Chromosomally mediated development of resistance is a well-recognized challenge of antipseudomonal therapy, occurring in over 10% of cases during active treatment.²⁰ Strains capable of “hypermutation,” which possess altered DNA mismatch repair systems and consequently exhibit mutation rates in excess of 1000 times those observed in wild-type isolates, have been identified in patients with cystic fibrosis and other chronic Pseudomonas infections. Major chromosomally encoded resistance mechanisms among Pseudomonas include AmpC beta-lactamase induction/derepression, porin channel mutations (particularly among the OprD porin channel), and efflux pump up-regulation.

The relatively impermeable membrane of Pseudomonas permits the passage of compounds into the cell through a group of over 100 barrel-shaped protein channels called porins. Porins serve an important physiologic function in promoting cellular uptake of sugars, peptides, and cations, but also allow the passage of hydrophilic antibiotics into
the cell. Loss of OprD porin from the outer membrane of P aeruginosa markedly increases the observed MICs to carbapenems, often conferring resistance to imipenem and pushing meropenem susceptibility into a clinically tenuous range. OprD down-regulation in P aeruginosa is mediated by a diverse molecular transcriptional apparatus that changes in response to growth conditions via quorum sensing.¹⁹

P aeruginosa possesses a wide array of drug efflux pumps, capable of actively expelling antibiotics from the cell after entry has occurred. Efflux pumps serve a variety of functions for P aeruginosa, exporting not only amphiphilic antibiotics from the intracellular compartment but also various environmental detergents and biocides. The most well established of these pumps is the MexAB-OprM efflux pump, which is able to export fluoroquinolones, tetracyclines, macrolides, trimethoprim, sulfonamides, and a variety of beta-lactams and BLIs, including carboxypenicillins, aztreonam, extended-spectrum cephalosporins, and meropenem.²¹

Fluoroquinolone resistance in P aeruginosa also emerges through chromosomal mutations in quinolone resistance determining regions (QRDRs). Mutations to the gyrA/gyrB genes (encoding DNA gyrase) and parC/parE genes (encoding topoisomerase IV) confer resistance more commonly to Pseudomonas than to Enterobacteriaceae, likely secondary to its inherently lower susceptibility. Emergent chromosomal mutations also appear to mediate rare cases of polymyxin resistance, through changes to the lipid A component of the bacterial outer membrane. Mutations in the regulatory apparatus controlling expression of lipid A synthetic enzymes in response to antimicrobial peptides have been well documented in polymyxin-resistant Pseudomonas strains.¹⁰

Resistance Acquired Through Mobile Genetic Elements Though chromosomally mediated mutations mediate a significant degree of P aeruginosa resistance, the role of acquired beta-lactamases is increasing. P aeruginosa possesses a conserved accessory genome that incorporates extrachromosomal elements such as plasmids, allowing for the horizontal transfer of beta-lactamases to clonal isolates. The most frequently acquired transferable betalactamases among P aeruginosa are PSE-1 and PSE-4, which confer modest resistance to many beta-lactams but lack clinically significant activity against selected extendedspectrum cephalosporins, aztreonam, and carbapenems. More problematic is the global emergence of acquired ESBLs and carbapenemases among high-risk Pseudomonas clonal isolates: PER-1, a geographically widespread Class A ESBL recognized in clusters of Europe; OXA-type ESBLs identified in outbreaks in Europe (particularly Turkey) and Southeast Asia; IMP- and VIM-type MBL carbapenemases identified in widespread fashion across the globe, often carried as cassettes within integrons, adjacent to aminoglycoside-modifying enzymes; and isolated reports of KPC-producing P aeruginosa.²²

Horizontal acquisition of mobile resistance determinants is also the primary determinant of aminoglycoside resistance among P aeruginosa. Drug-modifying enzymes capable of acetylation, adenylation, and phosphorylation of aminoglycosides have all been shown to confer high-level resistance to aminoglycoside agents, often in concert with up-regulated drug-efflux. The prevalence of rmtA genes, encoding a 16S rRNA methylase capable of modifying aminoglycoside target sites, has been notably increasing among clinical isolates.

Pseudomonas aeruginosa Clinical Epidemiology P aeruginosa originates primarily from environmental water sources and is an opportunistic pathogen; it is not a common commensal of the human intestinal microbiota but frequently colonizes patients with prior healthcare and antibiotic exposure. It historically has been associated with infections stemming from burn and trauma injuries but has since become a leading cause of nosocomial infection, currently accounting for 5% of all HAIs based on a large-scale 2015 nationwide point prevalence survey.²³ The predominant risk factors for infection due to carbapenem-resistant Pseudomonas aeruginosa (CRPA) are prior antibiotic use (particularly carbapenems), prolonged hospitalization or intensive care unit (ICU) stay, and indwelling medical devices. Other risk factors include chronic pulmonary disease (ie, cystic fibrosis) and immunocompromised state.²⁴^,²⁵

A meta-analysis of seven studies assessing attributable mortality of infections caused by CRPA found an increased risk of death compared to patients with carbapenem-susceptible P aeruginosa infection (pooled odds ratio 3.07, 95% CI, 1.60-5.89).¹⁵

Acinetobacter baumannii

Antimicrobial resistance among A baumannii has been steadily increasing over the last two decades, particularly in the nosocomial setting. Data from surveillance programs in the United States have demonstrated marked rises in Acinetobacter spp. resistance to extended-spectrum cephalosporins and quinolones, with rates exceeding 60% in 2009, and a more than doubling in rates of carbapenem (21%-48%) and colistin (3%-7%) resistance from 2003 to 2012.²⁶ Antimicrobial resistance in A baumannii is frequently observed in antibiotic treatment-experienced patients, and within ICUs and nursing homes (NHs). Infections caused by multidrugresistant Acinetobacter isolates are associated with higher rates of morality, in part because of a delay in initiation of effective antimicrobial therapy.²⁷

Non-Beta-Lactamase Resistance Mechanisms Acinetobacter spp. possess a relatively impermeable outer membrane, in part secondary to a low number of relatively small and impermissive membrane channels. Two major virulence factors embedded in the A baumannii envelope, LPSs and capsular exopolysaccharides, contribute to the organism’s inherent antimicrobial resistance. Capsular polysaccharides promote virulence and biofilm formation. LPS plays a major role in precipitating the inflammatory host response to A baumannii, and in vitro modifications to LPS can dramatically reduce colistin susceptibility.²⁷^,²⁸

Carbapenem resistance among Acinetobacter appears to be tightly linked to porin channels and outer membrane proteins. The Omp33-36 protein has been identified in A baumannii as a virulence factor, knockout of which enhances carbapenem resistance. Down-regulation or mutations to two membrane proteins, CarO and OprD, likely contributes to carbapenem resistance as well.²⁷^,²⁹

Relatively more is known about the role of drug efflux pumps in Acinetobacter antimicrobial resistance. Constitutive production and up-regulation of the AdeABC RND
efflux pump confers resistance to a number of beta-lactams and aminoglycosides and contributes to reduced susceptibility to fluoroquinolones. Similarly, overexpression of Tet(A), Tet(B), and AdelJK efflux pumps confers resistance to tetracyclines, one of the few remaining oral options for drug-resistant strains of Acinetobacter.²⁹

Alterations to antibiotic target sites also feature prominently in Acinetobacter antimicrobial resistance. Similar to other GNB, mutations in the GyrA (a subunit of DNA gyrase) and ParC (a subunit of topoisomerase IV) have been identified in fluoroquinolone-resistant (FR) isolates, as have mutations in dihydrofolate reductases in trimethoprim-resistant isolates. The efficacy of the BLI sulbactam, which possesses intrinsic antibacterial activity against A baumannii through the inhibition of penicillin-binding proteins PBP1 and PBP3, has been limited due to the emergence of PBP mutant isolates. Alterations to PBPs have also been implicated in imipenem resistance in selected strains of A baumannii.

Beta-Lactamase Resistance Mechanisms Acinetobacter spp. exhibit a remarkable genomic plasticity. A strain of Acinetobacter baylyi carries the ability to acquire 100-fold more exogenous genetic material via horizontal transfer than E coli. This “natural competency” is further evidenced by the relatively high frequency of foreign DNA noted in Acinetobacter genomic studies. Horizontally transferred genetic elements are accommodated by massive, chromosomal genetic resistance islands in Acinetobacter spp., harboring an impressive array of up to 45 genetic resistance determinants. These unique biologic characteristics of Acinetobacter have facilitated the emergence of a diverse array of beta-lactamases within high-risk clones, which are the primary drivers of the global epidemic of carbapenem resistance among A baumannii.

Carbapenem resistance among A baumannii is frequently mediated by oxacillinases, a diverse group of over 400 Ambler Class D beta-lactamases. Despite a lack of inherently robust carbapenem hydrolysis, OXA-type enzymes can precipitate clinical resistance to carbapenems by their synergistic interplay with other non-beta-lactamase resistance determinants or through enzyme hyperexpression. OXA-type carbapenemases are poorly inhibited by traditional BLIs (such as clavulanate and tazobactam) and are often ineffective against ceftazidime.⁸

The most prevalent OXA-type beta-lactamases in A baumannii are the OXA-51-like family of enzymes. OXA-51-like enzymes provide weak hydrolysis of carbapenems, often requiring contributions from other resistant determinants to precipitate frank resistance. OXA-23-like enzymes, on the other hand, are generally plasmid- or transposon-borne enzymes and are highly prevalent among carbapenem-resistant isolates. Owing to their ability to utilize a “hydrophobic bridge” with the substrates, they exhibit uniquely efficient carbapenem hydrolysis in comparison to other OXA-like enzymes and are often able to precipitate clinical resistance in the absence of other resistance mechanisms.

A baumannii possesses an intrinsic AmpC cephalosporinase with a similar spectrum of activity as in Enterobacteriaceae. Hyperexpression of AmpC beta-lactamases, often occurring as a result of promoter mutations, is frequently observed in multidrug-resistant isolates, conferring resistance to ceftazidime (a notable “hole” in the hydrolytic spectrum of many Acinetobacter oxacillinases). A wide spectrum of Ambler Class A beta-lactamases, including ESBLs (TEM- and SHV-derived, CTX-M family, PER family) and carbapenemases (GES and KPC enzymes) have been reported in Acinetobacter isolates, acquired through a variety of mobile genetic elements. Of emerging importance in A baumannii are the MBLs (VIM, IMP, SIM, and NDM), which have been globally observed in a series of high-risk clones.

Acinetobacter baumannii Clinical Epidemiology Acinetobacter species are particularly well adapted to the hospital environment based on their ability to survive for long periods on dry surfaces and can probably be transmitted via dust and fomites.³⁰ Primary risk factors for MDR A baumannii among hospitalized patients include critical illness, prior hospitalization, ventilator dependence, and prior antibiotic use.³¹ Critically ill patients with non-intact skin or ventilator dependence are at increased risk for colonization and A baumannii is a leading cause of VAP and is also associated with bacteremia, soft tissue infections, and meningitis. The ability of A baumannii to acquire multidrug resistance, as well as to survive on skin and in the environment, undoubtedly contributes to its success as a healthcare-associated pathogen.

Data from the U.S. NHSN in 2014 reported that 49.5% of A baumannii isolates in the United States were carbapenem resistant. High rates of carbapenem resistance are also found among isolates in Europe and Asia.³² The rising rates of carbapenem-resistant Acinetobacter baumannii (CRAB) over the past 15 years is predominantly due to the global dissemination of several multidrug-resistant clones.³³ CRAB infections typically occur in patients with prolonged hospitalization, particularly in ICUs. Risk factors include mechanical ventilation, trauma and burn injuries, multiple comorbidities, and prior antibiotic exposure.³⁴ Infection with CRAB is associated with a twofold increase in mortality compared to carbapenem-susceptible infections (OR 2.22, 95% CI, 1.66-2.98).³⁵

Stenotrophomonas maltophilia

Owing to inherent resistance to a number of antimicrobial agents including beta-lactams, S maltophilia is a nosocomial pathogen for which there are limited antimicrobial treatment options. Rates of quinolones resistance among S maltophilia have risen precipitously in recent years. While global rates of TMP-SMX susceptibility have remained >90%, reports of increasing TMP-SMX resistance have emerged, particularly among immunocompromised individuals and those with cystic fibrosis where resistance rates in excess of 30% have been noted.

TMP-SMX resistance within S maltophilia is mediated by mutations to dihydropteroate synthase (encoded by the sul1 and sul2 genes) and dihydrofolate reductase (encoded by the dhfr or dfrA generes). Such mutations are horizontally transmitted and frequently embedded within integrons among resistant S maltophilia isolates. Clonal expansion of such isolates has been observed globally.³⁶^,³⁷

The primary determinant of beta-lactam resistance in S maltophilia are the L1 and L2 chromosomal betalactamases. L1 is a MBL with a broad substrate spectrum including carbapenems. L2 is an Ambler Class A cephalosporinase with susceptibility to BLIs. These chromosomal beta-lactamases are constitutively expressed to varying degrees across S maltophilia strains but may also be selectively induced following antibiotic exposure.

Efflux pumps in S maltophilia are often basally expressed at low levels, but hyperexpression is observed in drug-resistant isolates secondary to mutations in their transcriptional regulatory apparatus. Hyperexpression of efflux pumps SmeDEF and SmeVWX appears to play a major role in quinolone resistance.

Stenotrophomonas maltophilia Clinical Epidemiology S maltophilia is another predominantly healthcareacquired pathogen that is also well adapted to wet environments. It is an opportunistic pathogen and is frequently associated with infections among critically ill and immunocompromised patients, particularly VAPs and CLABSIs. One single-center study found S maltophilia to be the fourth leading cause of VAP.³⁸ It is also a capable former of biofilms and can cause device-related infections. Several outbreaks have been associated with contaminated tap water sources such as sink drains.³⁹^,⁴⁰

Burkholderia cepacia Complex

The Burkholderia cepacia complex (BCC) encompasses at least 17 different organisms, the most prevalent of which are B cenocepacia and B multivorans. BCC causes significant morbidity and mortality, particularly among immunocompromised patients and those with cystic fibrosis, in part owing to its drug resistance mechanisms. BCC possesses a spectrum of chromosomal beta-lactamases including AmpC cephalosporinase and a PenB ESBL that is not responsive to BLIs. Both enzymes are coregulated through interactions with AmpR repression, cell wall degradation productions, and AmpD input in a manner analogous to that modeled in E cloacae. Some isolates harbor PenA, a BLI-resistant Class A carbapenemase with a similar hydrolytic spectrum to KPC-2. MBLs have also been observed in BCC isolates.⁴¹

As with other NF-GNB, efflux pumps play a primary role in antimicrobial resistance. Expression of the RND-3 in particular is a major mediator of fluoroquinolone and aminoglycoside resistance and appears to actively expel chlorhexidine during sessile growth phases. Importantly, polymyxins are not active against BCC, owing to a unique LPS structure that inhibits attachment to the bacterial outer membrane.⁴¹

Burkholderia cepacia Complex Clinical Epidemiology B cepacia complex are notorious respiratory pathogens among cystic fibrosis patients that are associated with pulmonary function decline and poor outcomes after lung transplantation. They are also environmental pathogens found predominantly in water sources and have been associated with outbreaks in non-cystic fibrosis populations stemming from contaminated medical equipment, particularly respiratory devices, as well as contaminated nonsterile water-based medications, such as saline flush syringes, liquid docusate, and chlorhexidine mouthwash.⁴²

RESERVOIRS AND MODES OF MDR-GNB TRANSMISSION

While it is important to approach the epidemiology of MDR Gram-negative pathogens from an organism-specific perspective, it is also important to consider potential reservoirs and modes of transmission for MDR-GNB as a whole. Mention will be made of specific organisms with regard to unique characteristics pertaining to reservoirs and modes of transmission.

Patients and Healthcare Personnel

As with other multidrug-resistant organisms (MDROs), patients colonized or infected with MDR-GNB serve as the primary reservoir and source of patient-to-patient transmission in most inpatient healthcare settings.⁴³ Hands of healthcare personnel (HCP) can become transiently contaminated with MDR-GNB in the course of routine clinical care with potential to transmit pathogens to other patients in the absence of appropriate hand hygiene. This problem is magnified by factors such as increased MDR-GNB colonization pressure and reduction in hand hygiene compliance, both of which increase the probability of MDR-GNB spread. A study by Rock et al. found that HCP caring for patients infected or colonized with KPC-producing K pneumoniae contaminated their hands or gloves after 10.4% of encounters.⁴⁴ Morgan et al. found that HCP gowns or gloves were contaminated after 33% of interactions with patients colonized with MDR A baumannii and after 17% of encounters with patients colonized with MDR P aeruginosa.⁴⁵ With high rates of HCP contamination occurring during patient care, hand hygiene noncompliance can result in transmission to patients by the HCP. Grabowski et al. performed a case-control study comparing patients who acquired CRE colonization vs those who did not and found that patients were at increased risk of CRE acquisition if they shared ≥1 healthcare provider with other CRE patients.⁴⁶ A study utilizing a mathematical modeling program compared the effectiveness of incremental improvements in hand hygiene vs environmental cleaning. Improvements in hand hygiene compliance was determined to have twice the impact of an equivalent improvement in environmental cleaning on the reduction of A baumannii infections.⁴⁷

Hospital Environment

The hospital environment serves as a potential source of MDR-GNB acquisition. MDR-GNB have been found in virtually every moist area of the hospital, including in various types of fluids, and on an array of equipment and surfaces that were exposed to the hands, secretions, and/or excretions of patients. Inanimate objects in the patient room can become contaminated with MDR-GNB due to shedding by the colonized patient. These pathogens can then be transmitted to other patients primarily by contamination of the hands of HCP in the setting of suboptimal hand hygiene. Patient acquisition of MDR-GNB can lead to invasive infection in patients in high-acuity care settings such as ICUs, due to frequent use of invasive devices and high degree of comorbid conditions.⁴⁸

A baumannii is the MDR-GNB most strongly associated with outbreaks due to environmental contamination. Acinetobacter has been cultured from the skin of one-fourth of nonhospitalized individuals and about one-third of hospitalized patients, most commonly from intertriginous areas such as toe webs and the groin. A baumannii thrives in humid conditions but is also capable of surviving on dry surfaces for up to 5 months.⁴⁹ It has been found to frequently colonize HCP hands and uniforms.⁵⁰ Contamination of ambient air from patients with oropharyngeal or rectal A baumannii colonization has also been described.⁵¹

Prior outbreaks of MDR A baumannii have previously been frequently associated with contaminated dry hightouch surfaces such as bedside tables, bed rails, and curtains.⁵² Other recognized colonized environmental sources include ventilators, sinks and suction equipment. Inadequate postdischarge cleaning of a patient’s room who was colonized or infected with A baumannii can lead to the acquisition of the same GNR-MDR by subsequent patients in that room. One study found that risk of MDR A baumannii HAI was 4 times higher for patients admitted to a room where a prior occupant was colonized with MDR A baumannii (OR 4.2, 95% CI, 2-8.8, P < .001).⁵³

A prospective screening study of P aeruginosa upon admission to an Italian ICU (oropharynx, rectal, nasal, and axillary swabs) determined that, based on identical pulsed field gel electrophoresis (PFGE) patterns, 25/42 (59.5%) episodes of hospital-acquired P aeruginosa infection or colonization were due to cross transmission.⁵⁴ Risk of MDR P aeruginosa HAI has also been associated with admission to a room where a prior occupant was colonized with MDR P aeruginosa, though the strength of this association is less than for A baumannii (OR 2.3, 95% CI, 1.2-4.3, P = .012).⁵³ P aeruginosa outbreaks often occur among immunocompromised and hematology/oncology patients. Sources of MDR P aeruginosa HAI have most been linked to contaminated devices and water sources.⁵⁵

CRE also have the capacity to contaminate the hospital environment. A study of 34 patients who were colonized or infected with KPC-producing K pneumoniae found cultures positive for CPKP in the immediate surroundings of 88% of patients.⁵⁶ Commonly contaminated sites included the floor, bed frame, furniture, and bedsheets. Investigation of an outbreak of KPC-producing K pneumoniae at the National Institute of Health utilizing whole genome sequencing implicated a mechanical ventilator contaminated with a strain of KPC K pneumoniae, despite the ventilator having undergone multiple rounds of disinfection with quaternary ammonium and bleach products.⁵⁷

Water Sources

Hospital wastewater has been increasingly recognized as a GNB reservoir, as GNB are particularly well-suited to survive in moist environments. Several factors make hospital water systems a suitable environment for bacterial persistence, such as complex plumbing systems, potential for areas of water stagnation, particularly sink traps, and frequent exposure of sinks to contaminated fluids during routine care of MDR-GNB colonized patients. Furthermore, many MDR-GNB readily form biofilms on wet inanimate surfaces, making chemical disinfection challenging. Multiple outbreaks have been associated with contaminated sinks in wards caring for immunocompromised populations.⁵⁸ Splash water or aerosols generated from sinks harboring MDR-GNB can then contaminate the surrounding room structures as well as the hands of HCWs and lead to patient-to-patient transmission.

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