Continuing Education Activity

Gram-negative bacteria (GNB) are among the world's most significant public health problems due to their high resistance to antibiotics. These microorganisms have significant clinical importance in hospitals because they often require patients to be in the intensive care unit (ICU), and patients are at high risk of morbidity and mortality. Two large groups, Enterobacteriaceae and the non-fermenters, are responsible for most clinical isolates; nevertheless, other clinically pertinent gram-negative organisms exist, including Neisseria, Haemophilus spp., Helicobacter pylori, and Chlamydia trachomatis. This activity reviews the evaluation of gram-negative bacteria and the interprofessional team's role in managing patients with this condition.

Objectives:

• Outline the types of gram-negative organisms that are clinically significant.
• Identify the treatment options for gram-negative infections based on the location and type of infection.
• Describe the mechanisms by which gram-negative bacteria can become resistant to antibiotics.
• Explain modalities to improve care coordination among interprofessional team members to improve outcomes for patients affected by gram-negative bacteria.

Introduction

Gram-negative bacteria (GNB) are among the world's most significant public health problems due to their high resistance to antibiotics. These microorganisms have significant clinical importance in hospitals because they put patients in the intensive care unit (ICU) at high risk and lead to high morbidity and mortality.[1][2] Two large groups, Enterobacteriaceae and the non-fermenters, are responsible for most clinical isolates; nevertheless, other clinically concerning gram-negative organisms exist, including but not limited to Neisseria, Haemophilus spp., Helicobacter pylori, and Chlamydia trachomatis. 

Enterobacteriaceae 

Enterobacteriaceae are a heterogeneous group widely dispersed in nature. They account for about 80% of gram-negative isolates with a myriad of disease-causing general/species in humans, including urinary tract infections, pneumonia, diarrhea, meningitis, sepsis, endotoxic shock, and many others. The general/species that frequently affect humans are Escherichia, Proteus, Enterobacter, Klebsiella, Citrobacter, Yersinia, Shigella, and Salmonella, among others. Laboratory characterization is an essential component when it comes to microorganisms; therefore, it is imperative to expose characteristics of Enterobacteriaceae, which are bacilli, non-sporulated, have variable motility, grow in the presence and absence of oxygen, ferment organisms of glucose, are cytochrome oxidase negative, and can reduce nitrate to nitrite.[3]

Non-Fermenters

The non-fermenter, gram-negative bacilli (BNF) have a lower frequency of isolation when compared to Enterobacteriaceae; however, they are a relevant group since they cause severe, fatal infections, especially in the hospital environment. They also cause opportunistic diseases in ICU patients who undergo invasive procedures. The main BNF microorganisms that cause human disease are Pseudomonas aeruginosa, Acinetobacter baumannii, Burkholderia cepacia, Burkholderia pseudomallei, Stenotrophomonas., Alcaligenes, and Moraxella. These stand out for being aerobic and non-sporulated; they are incapable of fermenting sugars, using them through the oxidative route.[4]

The critical issue regarding BNF, when it comes to the antimicrobial sensitivity profile, is undoubtedly their intrinsic resistance since they produce a variety of genes with multiple mechanisms capable of mitigating the microbicidal action. Thus, it stands out in P. aeruginosa, cephalosporinase of type AmpC, and efflux systems that confer resistance to b-lactams. The most frequent are MexAB-OprM; and loss of OprD (which gives impermeability to the bacterial cell due to the loss of porin.)[5][6] Acinetobacter baumannii naturally produces AmpC cephalosporinase and oxacillinase (OXA), leaving it spontaneously immune to many drugs. The genetic ingenuity of this microorganism goes further, and it combines high impermeability with genetic plasticity, combining with the resistance of mechanisms such as extended-spectrum b-lactamases (ESBL).[7] The Stenotrophomonas exhibit a pattern of intrinsic multi-resistance, especially in patients who have had contact with carbapenems. Thus, Stenotrophomonas present several efflux pumps and produce two carbapenemases – L1 (resistance to all carbapenems) and L2 (cephalosporinase). [8] These mechanisms, associated or separate, restrict the treatment options to an alarming level. Sulfamethoxazole-trimethoprim remains the mainstay of treatment.[9] 

Antibiotic Resistance

These organisms have a range of mechanisms to prevent the action of many antimicrobials used in clinical medicine. Some of the mechanisms of resistance include efflux pumps,[10] alteration of the drug binding site and membrane permeability,[11] degradation enzymes, and the conformational change of the drug culminating in its inactivation.[12]

GNB have two membranes, an external and an internal. The external membrane expresses a potent immune response inducer, lipopolysaccharide (LPS), which is composed of three units: a hydrophilic polysaccharide, O antigen, and a hydrophobic domain known as lipid A. Lipid A are responsible for the higher endotoxic activity of these bacteria.[13][14] However, the LPS is heterogeneous in the various bacterial groups, and some bacteria manifest this antigen weakly due to genetic changes and are not recognized by Toll-like receptors. In contrast, there are BGN groups that can trigger such a response in large proportions. Thus, LPS can trigger the innate immune response through Toll-like receptors 4 (TLR4), which occurs in many immune cells such as monocytes, macrophages, dendritic cells, and neutrophils. The resulting activation of the innate immune response mediated by LPS together with TLR4 receptors culminates in an exacerbated response with the production of cytokines, chemokines, and interferons and their suppression.

Enterobacteriaceae diffuse their plasmids by conjugation, which gives rise to resistance to almost all existing antibiotics. The family of enzymes carbapenemase – KPC, NDM-1, IMP, VIM, OXA-48 – is undoubtedly one of the most significant health challenges of the century, given the potential for dissemination between species and mortality rates due to infections caused by bacteria with such plasmids.[15] Colistin, one of the few antibiotics that still treat multiresistant infections, already has a mobile resistance gene, mcr-1, and Enterobacteriaceae has a crucial role in the spread of this gene, with worldwide reports.[16] Moreover, a further concern is that they usually associate these genes with other resistance genes (CTX-M, NDM, IMP), producing resistance to cephalosporins and carbapenems, enhancing the deleterious effects caused by these microorganisms.[13]

Etiology

The gram-negative bacteria have a great ability to cause disease in humans and can reach almost all systems in the organism, such as the digestive system, nervous system, urinary system, and bloodstream, causing diarrheal gastroenteritis until severe meningitis. Such microorganisms colonize the intestines, airways, and skin, which favors the spread to other parts of the human organism, especially in immunocompromised individuals. One of the greatest difficulties of health professionals is to treat nosocomial infections of the lower respiratory tract in which the pathogens GNB are involved because although they are responsible for a good portion of these infections, they are non-responsive to antibiotic therapy due to the high resistance rates and the poor penetration of drugs into the lung parenchyma.[17] 

Another major concern is gastroenteritis caused by Enterobacteriaceae (Shigella spp., Salmonella spp., enteropathogenic E. coli), which affects millions of people worldwide and is related to a lack of sanitation.[18] Additionally, they are responsible for meningitis - a potentially fatal disease if not treated in time – it is acquired both in the community and the hospital environment.[19][20] Urinary tract infections are also common, especially in young women. However, these infections became a problem with the rampant emergence of multi-resistant bacteria. Finally, bacteremia is an important complication of these infections because of the resistance mentioned above that the microorganisms demonstrated.[19]

Epidemiology

Considering the cosmopolitan character of gram-negative microorganisms and their ability to cause infections in humans, it is important to state that the biggest problem facing healthcare workers is bacterial groups that cause multiresistant infections. Multiresistant gram-negative infections (MDRs) are today one of the most significant health challenges in the world due to the inadequate response of these pathogens to antimicrobials, which have practically pulverized by the production of ESBL and carbapenemases. A little over twenty years ago, the first KPC carbapenemase was reported in the United States, and since then, such infections have spread globally. However, since the 1980s, there have been reports of gram-negative strains of ESBL, especially in the hospital environment.[20] 

According to the Centers for Disease Control and Prevention (CDC), multiresistant gram-negative bacteria are rampant in the United States except in Maine and Idaho.[20] Still, in the US, Livorsi et al. found a variation in the incidence from 0.3 to 2.93 infections per 100000 person-years. Outside the United States, there are already reports of multiresistant bacteria on almost every continent. In Europe, for example, about 25000 people die each year from multidrug-resistant (MDR) infections.[21]

The extended-spectrum beta-lactamases (CTX-M, TEM, SHV, PER, VEB, TLA) are enzymes that hydrolyze extended-spectrum cephalosporins. From this family of enzymes, the most prevalent are CTX-M and its variations. Studies show that 12% of E. coli isolates in the USA produce ESBL, while in Latin America and Asia, this percentage may reach 27% and 38%, respectively. At the beginning of the ESBL outbreaks, they had links to the hospital environment; however, today, they are found in patients originating in the community.[22][23] 

Carbapenemases (KPC, NDM-1, IMP, VIM, OXA-48) are characteristically enzymes that hydrolyze carbapenem and most other beta-lactams. In the United States, the most commonly detected are KPC, NDM, and OXA-48; in Europe, the most prevalent are OXA-48, KPC, and VIM, and the NDM are less incident. Further, an aggravation of these enzymes is their transfer by plasmids, which often carry additional resistance genes, making these gram-negative bacteria pan-resistant.[22]

Finally, another vector of the global spread of these genes is the extensive capacity of interspecies transmission. Also, it is postulated that there is a risk of contracting a multidrug-resistant infection in patients with comorbidities, hospitalizations in ICUs, prior use of antibiotics, and prolonged use of a central venous catheter.[20]

History and Physical

The findings of gram-negative infections are non-specific, not distinguishing them from other infectious diseases on physical examination. A classic example is pneumonia; its symptoms include fever, chest pain, dyspnea, and purulent expectoration; these symptoms may correlate with tachycardia, tachypnea, hypoxemia, and auscultatory signs of consolidation. However, these signs are not specific to the disease. Cultures are the only way to determine the organism.[24] Similarly, in bacterial meningitis, one may note fever, severe headache, nausea, vomiting, neck stiffness, prostration, and mental confusion. However, such features are not exclusive, requiring other evidence to conclude the type of organism.[25]

Evaluation

Whereas the infections produced by gram-negative are practically indistinguishable from other etiologies, laboratory characterization can define the organism and its antimicrobial susceptibility profile. Thus, the gold-standard examination of these infections is the cultures; however, they have the problem of a delay in the result. Another essential exam for bacterial isolates is the Gram stain; although simple, it can quickly distinguish the course of a drug intervention. Because of their heterogeneity, Enterobacteria depend on various biochemical tests (Indol, Voges-Proskauer, catalase, cytochrome oxidase, urease, motility, citrate, ONPG, Dnase, decarboxylation of lysine, gas production, among others), grown in primary isolation agar. Species such as E. coli, because they are fermenters of strong acids, they produce large quantities of mixed acids, which make the pH of the media present pink colonies. Others, such as non-fermenters, grow in primary isolation media (blood agar and MacConkey) in the OF-oxidation/fermentation medium - grow only in the aerobic environment.[3]

However, it is essential to mention that identifying bacterial strains resistant to multiple drugs may require molecular methods, but they are not available in all laboratories. Phenotypic methods such as Modified Hodge (MHT) and Combined Diffusion Disc (CDT) using EDTA are good alternatives.[26] The MHT is a test based on the inactivation of carbapenems by bacterial strains containing the enzymes carbapenemases, enabling a susceptible strain to extend its growth to a disc containing the antimicrobial along the inoculum of the strain tested. This test is recommended for strains with high minimum inhibitory concentration (MIC) or reduced zones of inhibition in the disk.[27] The CDT comprises a test disc-diffusion with carbapenem antibiotics, where carbapenem discs are placed with and without EDTA. Their connection predicts whether the organism produces or does not carbapenemase.[28]

Treatment / Management

Treatment options for gram-negative MDR infections are limited, and the results are generally disappointing because of drug resistance. MDRs are still evolving, which provides resistance to novel antimicrobials. Thus, as new drugs' availability progresses slowly, some previously abandoned options have reappeared, such as polymyxins and colistin, which have high toxicity (nephrotoxicity and neurotoxicity). Furthermore, resistant genes to these drugs have been reported, such as mcr-1 - which causes additional concern. But it is known that combining these drugs with carbapenems may have an improved synergistic action.[29] Another drug of choice is tigecycline since it shows in vitro activity against MDR; however, there are limitations in its use as high doses are required, and tissue penetration is often poor, which impairs its action in vivo.[30]

Fosfomycin, an old drug used to treat urinary tract infections, has also resurfaced as a potential agent for treating these MDR infections. However, monotherapy with this drug also results in resistance, and its use is appropriate in conjunction with other agents such as carbapenems and polymyxin. This drug has moderate absorption, restricting its use only for urinary tract infections.[31] Drugs once rejected because of their nephrotoxicity and ototoxicity, the aminoglycosides, have resurfaced again because of their effectiveness against gram-negative organisms. However, these drugs do not perform the same against MDRs except for urinary tract infections; they also exhibit less toxicity compared to polymyxin and tigecycline and may be useful in combination therapy.[32]

The carbapenems are used for severe infections secondary to ESBL-producing organisms; however, the appearance of carbapenemases has made treatment difficult. Monotherapy can be effective. Combination therapy with these drugs is often effective because of the high affinity of carbapenemases for ertapenem, making it a "bait" for the action of other carbapenems.

New drugs such as ceftazidime-avibactam and meropenem-vaborbactam have emerged; the first is an antipseudomonal cephalosporin associated with a beta-lactamase inhibitor, while the second is a carbapenem with b-serine and has anti-lactamase activity. Early results are encouraging. [20] Ceftazidime-avibactam has activity against ESBLs, AmpC-, KPC, and OXA-48-producing pathogens, as well as drug-resistant Pseudomonas aeruginosa isolates. It does not have activity against metallo-β-lactamase-producing strains.[33] A combination of aztreonam and ceftazidime-avibactam could be active against serine-β-lactamase- and metallo-β-lactamase-producing P. aeruginosa.[34] The combination also showed significant synergy in most NDM-producing Enterobacterales, where they were resistant to aztreonam and ceftazidime-avibactam separately.[35]

Cefiderocol is a novel injectable siderophore cephalosporin that combines a cephalosporin core and a catechol-type siderophore with side chains similar to ceftazidime and cefepime. This structure and mechanism of action confer increased stability against numerous β-lactamases, including ESBLs such as CTX-M and carbapenemases such as NDM, KPC, VIM, IMP, OXA-23, 48-like, 51-like, and 58. It is active against both lactose-fermenters and non-fermenters.[36] Colistin is another alternative, but its use is again limited by high adverse effects and increasing resistance.[37]

Differential Diagnosis

• Gram-positive bacterial infections (Staphylococcus spp., Streptococcus spp., Micrococcus spp.)
• Viral infections (HIV, respiratory syncytial virus, influenzas)
• Mycobacterium spp. and Nocardia spp.
• Fungal infections (Cryptococcus spp, Aspergillus spp., Candida albicans)

Toxicity and Adverse Effect Management

Polymyxins have been in disuse since the 1970s because of their known toxicity, mainly because of their nephrotoxic effects. With the appearance of gram-negative MDR infections, they have been revitalized. However, there are reports of problems with toxicity and increased resistance to these drugs.[38] The mechanism of renal toxicity exerted by these drugs occurs mainly through oxidative stress, resulting in mitochondrial dysfunction and loss of the membrane potential of this organelle. Apoptosis is also another effect attributed to this drug, which activates caspases 3, 8, and 9 of pulmonary epithelial cells.[39] The toxicity of polymyxins is related to concentration; however, the current pharmacokinetics and pharmacodynamics have already shown the dosage for this class of drugs is not the most appropriate, thus requiring new studies for correction, which could attenuate the toxicity of the drug.

Another class of drugs with toxic potential is aminoglycosides which mainly cause ototoxicity and nephrotoxicity. These drugs block cationic channels of the ciliated cells in the inner ear. They have the same action as the mechanosensitive transduction channels of these cells; once inside these cells, they promote biochemical changes that culminate in the elevation of intracellular calcium and the formation of reactive species of oxygen (EROS), which irreversibly damage the cell.[40] The nephrotoxicity of the aminoglycosides is mainly related to damage to the proximal tubule; the effects are relative to the dose and the time of exposure. The endocytosis of these drugs with consequent release of the lysosome's content is the mechanism by which the drugs exert their toxicity; they also block the calcium channels, culminating in the loss of this ion, magnesium, and potassium.[41]

Beta-lactams, including penicillins, cephalosporins, and carbapenems, as well as beta-lactam plus beta-lactamase inhibitor combinations, are frequently associated with drug allergy, but cross-reactivity rates are variable.[42] Other adverse effects include predominantly gastrointestinal effects, including diarrhea and abdominal pain. [43]

Prognosis

Many variables are present in gram-negative infections; therefore, the prognosis of the diseases caused by these pathogens is difficult to measure and is not unanimous in the literature. However, such infections are usually associated with poor outcomes, mainly in elderly patients, with a history of comorbidities, those with solid organ transplantation, and malignant diseases. Treatment success also depends on the causative microorganism and the site of infection. Serious infections with high mortality rates are related to delayed antibiotic therapy or inadequate therapy, while appropriate antibiotic administration correlates with patient survival. Besides, the lack of a well-defined protocol for these infections may lead to treatment failure since suboptimal or inadequate administration contributes to poor results. Finally, monotherapy is also ineffective, especially when compared to combination therapy, which is related to a good prognosis.[44]

Complications

Several complications may result from gram-negative infections, especially the enzyme producers that hydrolyze carbapenems. A concerning infection due to recurrence and ease of acquisition is the urinary tract, as it is increasingly common to find multidrug-resistant organisms in the community, and such infections, if not treated properly, could lead to renal failure, sepsis, and even death.[19] Similarly, nosocomial infections in frail patients with suppressed immunity, comorbidities, and transient immunosuppressive status make this combination challenging for health professionals.[20]

In this sense, special care is necessary for patients who have suffered burns because there are more ports of entry for microorganisms; infections in burn patients can be potentially lethal and invasive depending on the microbial load and the pathogen itself.[45] Another risk is respiratory tract infections, mainly associated with mechanical ventilation, since gram-negative bacteria are responsible for such infections, with Enterobacteriaceae and non-fermenters involved, with considerable potential for fatality.[46]

Deterrence and Patient Education

One of the chief difficulties in treating multiresistant gram-negative infections is the excessive use of antibiotics, not only those agents acquired in the community but also in hospitals. As the MDRs became epidemic, it became apparent that overuse of these antimicrobials was one of the causes that urgently required attention. Thus, measures aimed at changing the use of these drugs, with educational campaigns, as well as the fight against self-medication with practices such as monitoring drug consumption and their registration in pharmacies, are necessary to change the habits of the population and also health professionals.

Enhancing Healthcare Team Outcomes

The problem of multiresistant GNB infections has been rising since the beginning of the 21st century. However, there is difficulty in eradicating drug-resistant organisms because of the lack of effective antibiotics. Thus, simple measures such as regular hand hygiene, adequate sterilization of medical equipment, and isolation of patients suspected or diagnosed with MDR microorganisms, especially those submitted to invasive procedures, with the staff using caution regarding the entry of external persons. All individuals should abide by infectious disease preventive rules established in the healthcare setting. Another significant action is the laboratory worker who isolates MDR pathogens to immediately inform the epidemiological surveillance staff of the health establishment so that prevention and control measures are enacted promptly.

In summary, the results at present are still not good, especially in elderly patients with a history of multiresistant GNB infection; however, the measures above, although simple, could save lives as well as avoid the spread of these MDR bacterial strains, which pose a high risk to society for the reduced spectrum of drugs that address these infections, which, in addition to being often inefficient, also present high levels of toxicity. [Level 1]

Given the problems of multi-drug resistant bacteria, fighting Gram-negative infections requires an interprofessional healthcare team approach. This can include family clinicians, infectious disease specialists, nurses, and pharmacists, some of whom will also be board-certified in infectious disease. All team members must keep accurate records of their interactions and interventions with these patients, so everyone on the interprofessional team can access the same updated patient data and make the appropriate decisions. Open communication lines between all members are also crucial so that each member can contact anyone from other disciplines to ensure the patient gets the best possible care. [Level 5]