Multi-drug resistant gram-negative bacterial pneumonia: etiology, risk factors, and drug resistance patterns.

Bacterial pneumonia is one of the most serious public health issues owing to its medical and economic costs, which result in increased morbidity and mortality in people of all ages around the world. Furthermore, antimicrobial resistance has risen over time, and the advent of multi-drug resistance in GNB complicates therapy and has a detrimental impact on patient outcomes. The current review aimed to summarize bacterial pneumonia with an emphasis on gram-negative etiology, pathogenesis, risk factors, resistance mechanisms, treatment updates, and vaccine concerns to tackle the problem before it causes a serious consequence. In conclusion, the global prevalence of GNB in CAP was reported 49.7% to 83.1%, whereas in VAP patients ranged between 76.13% to 95.3%. The most commonly reported MDR-GNB causes of pneumonia were A. baumannii, K. pneumoniae, and P. aeruginosa, with A. baumannii isolated particularly in VAP patients and the elderly. In most studies, ampicillin, tetracyclines, amoxicillin-clavulanic acid, cephalosporins, and carbapenems were shown to be highly resistant. Prior MDR-GNB infection, older age, previous use of broad-spectrum antibiotics, high frequency of local antibiotic resistance, prolonged hospital stays, ICU admission, mechanical ventilation, and immunosuppression are associated with the MDR-GNB colonization. S. maltophilia was reported as a severe cause of HAP/VAP in patients with mechanically ventilated and having hematologic malignancy due to its ability of biofilm formation, site adhesion in respiratory devices, and its intrinsic and acquired drug resistance mechanisms. Effective combination therapies targeting PDR strains and drug-resistant genes, antibiofilm agents, gene-based vaccinations, and pathogen-specific lymphocytes should be developed in the future.

Introduction

Pneumonia is an acute inflammation and consolidation of lung tissue due to infectious agents such as bacteria, viruses, fungi, and parasites [1]. Bacterial pneumonia is an inflammation of one or two lobes of the lung due to bacterial infection [2]. Based on how the infection is acquired, pneumonia can be classified into community-acquired pneumonia (CAP) and hospital-acquired pneumonia (HAP) [3]. According to Temesgen and his colleague’s report in 2019, CAP is an infection of the lung parenchyma that is not acquired from a hospital or health care facility [4]. Hospital-acquired pneumonia is defined as pneumonia that occurs after 48 h or more of hospital admission, and if associated with mechanical ventilation, it is termed ventilator-associated pneumonia (VAP) [5]. The global burden of diseases, injuries, and risk factors study in 2017 reported that lower respiratory tract infections (LRTIs), including bacterial pneumonia, cause nearly 2.56 million deaths among all age groups, making LRTIs the fifth leading cause of mortality with higher fatalities in Sub-Saharan Africa, South Asia, and Southeast Asia [6, 7].

Bacterial pneumonia can spread via aspiration, inhalation, or bloodstream spread of pathogenic bacteria [8]. Pneumonia is a result of an infection caused by the immune system’s inability to clear a pathogen from the lower airway and alveoli. This leads to the bronchioles and alveoli being filled with inflammatory exudates of leukocytes and fluid. This results in decreased carbon dioxide and oxygen exchange between the blood and the lungs, causing respiratory scarcities and symptoms such as cough, sputum production, dyspnea, chest pain, and respiratory dysfunction and/or shock in severe cases [9, 10]. According to several studies, age, incomplete or inadequate vaccination, indoor environmental exposure, medical conditions such as asthma, diabetes, heart disease, treatment-induced cytopenias in cancer, long-term hospitalization, malnutrition, immunosuppression, smoking, alcohol consumption, poor dental hygiene, contact with contaminated hospital equipment, previous exposure to antibiotics, and the presence of viral infections that compromise the respiratory tract that results in secondary bacterial colonization and infection are all important risk factors for disease development [11-15].

Studies documented that Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, Moraxella catarrhalis, and Escherichia coli were the most frequent causes of typical pneumonia, whereas atypical pneumonia is mostly caused by Legionella pneumophila, Chlamydia pneumoniae, and Mycoplasma pneumoniae. Even though S. pneumoniae is the most prevalent cause of CAP in all age groups around the world, gram-negative bacteria (GNB) such as K. pneumoniae, Acinetobacter baumannii, P. aeruginosa, and E. coli are commonly related to HAP [3, 16, 17]. Antibiotic resistance is increasingly being recognized as a major worldwide health concern resulting from antibiotic overuse and improper administration [18]. Nowadays, pneumonia caused by multidrug-resistant gram-negative bacteria (MDR-GNB) is growing more common and has a detrimental impact on patient outcomes, indicating a shift in infection trends to GNB and their rapid dissemination, particularly in the hospital settings [19-23]. To address this, the current review is intended to provide a summary of the findings on bacterial pneumonia focusing on gram-negative etiology, their pathogenesis, mechanisms of resistance to antibiotics, risk factors, diagnostic challenges and advancements, updates on treatment options, and vaccine issues which enables concerned bodies to tackle the problem before it causes a serious consequence.

Epidemiology and burden

Bacterial pneumonia continues to be one of the most serious public health problems due to its medical and economic burden. Both CAP and HAP increase morbidity and death in people of all ages around the world [24, 25]. Community-acquired pneumonia is the sixth leading cause of death in people aged 65 and above worldwide. In developed countries, the estimated incidence of CAP is 0.2 to 1.1% in adults, and the mortality is 2 to 14% [26]. A population-based study by Bjamason et al. [27] reported that CAP requiring hospitalization was 2.7 cases per 1000 adults annually. The incidence is higher in children under the age of four and people over the age of 60, with more than 12 cases per 1000 people, but in adults, the rate is usually 5.2 to 7.1 cases per 1000 persons per year [28]. Childhood mortality and adult hospitalization due to pneumonia remain increasing in low and middle-income countries. The frequency has increased in the elderly because of physiological changes linked to the progressive dysfunction of the respiratory tract and/or weakened immunity supported by 72.6% GNB prevalence in elderly patients with CAP from China [26, 29].

According to hospital-based studies in Africa, CAP is linked to a 6 to 15% increase in adult inpatient hospital mortality [30]. Community-acquired pneumonia is the most common cause of adult hospitalization and mortality, accounting for 10% in Kenya [31], 11.9% in Nigeria [32], 17% in Ethiopia [33], and 51,000 admissions with 10,000 deaths in Malawi [34] each year. Epidemiological reports from Sub-Saharan Africa also revealed high rates of morbidity and mortality from the disease, with an estimated 4 million cases and 200,000 deaths per year [35, 36]. Pneumonia is one of the leading causes of death among Ethiopian children under the age of five, accounting for 28% of all deaths [33]. Previous reports in Africa showed GNB prevalence in CAP with 49.7% to 56.7% in Ethiopia [4, 37, 38], 83.1% in Tanzania [39], and 76.2% in Sudan [40].

Hospital-acquired pneumonia is the second most common nosocomial infection in the world, affecting 0.5 to 1.7% of hospitalized patients. It is also the leading cause of death among all nosocomial infections [41]. The incidence of HAP ranges from 5 to 20 or more cases per 1000 hospital admission [42]. A study in Ethiopia by Tassew et al. [43] in hospitalized patients reported HAP as the common type of infection, accounting for 24.7%. Ventilator-associated pneumonia is the most common nosocomial infection in the intensive care unit (ICU), accounting for 25% of all ICU infections [44]. A Kenyan study reported VAP prevalence of 54.4% among 92 patients with clinical pulmonary infection [45]. In a large cohort study, both HAP and VAP in ICU patients were associated with 82% and a 38% increase in the risk of 30-day mortality, respectively [46]. Gram-negative bacteria are responsible for most bacterial causes of HAP/VAP (50–80%) [47]. In the US and Europe, HAP and VAP due to GNB among ICU patients were 61.5 and 76.1%, respectively [48]. A study conducted on Egyptian children revealed that GNB was more prevalent in HAP and VAP (91.67 and 87.8%, respectively) [49]. In Iran, GNB was obtained in 72.2 and 84.6% of HAP and VAP, respectively [50]. Feng et al. [51] reported a 14.5% mortality rate of HAP related to GNB in a retrospective, single-center analysis study in China. Moreover, MDR, extensively drug-resistant (XDR), and pan-drug resistant (PDR) bacteria, especially GNB, are increasingly isolated in HAP and VAP and are associated with mortality rates over 50% [52].

Etiology

Recently, several studies on the bacterial cause of pneumonia have been published. Studies reported the study period, the number of study participants, age category, pneumonia type (CAP, HAP, and VAP), specimen sources, as well as studies performed culture and antimicrobial susceptibility testing have been included and summarized (Tables 1 and 2). In studies conducted on the etiology of CAP, GNB was found to be present 49.7 to 83.1% of the time, with common etiologic agents of K. pneumoniae, P. aeruginosa, and E. coli [4, 26, 37–40] (Table 1). The very high prevalence of GNB in some studies is related to variation in the sample size, geographic location, study period, study population, and specimen contamination of respiratory flora. Ventilator-associated pneumonia caused by multidrug-resistant GNB has emerged as a significant and intractable clinical problem [58]. Studies in VAP patients reported that GNB prevalence between 76.13 to 95.3% with highly MDR P. aeruginosa, and A. baumannii strains [53-57] (Table 2).

Table 1

Summary of the isolation and drug resistance profile of GNB in CAP

Country	Study year	Participants, age group, pneumonia category	Specimen	GNB (%) in culture	Frequently isolated GNB (%)	Decreasing order of resistance, MDR (%)	References
Tanzania	2015	353, adult, CAP	Sputum	83.1%	K. pneumoniae (29.9%) and P. aeruginosa (11.7%)	K. pneumoniae: AMP > AMC > CRO P. aeruginosa: AMP/SXT/AMC > CRO > CIP	[39]
China	2016 to 2017	176, older (>  60 years), CAP	Sputum	72.6%	K. pneumoniae (27.4%), E. coli (17.9%), and P. aeruginosa (10.3%)	K. pneumoniae: AMP > CXM > PIP, 25.0% ESBL E. coli: AMP > CXM > CTX > PIP, 42.9% ESBL P. aeruginosa: MIN > SXT > CXM	[26]
Sudan	2017	100, 16 to 60 years, CAP	Sputum	76.2%	K. pneumoniae (42.8%) and P. aeruginosa (30.9%)	K. pneumoniae: CAZ/PEP/CRO/CTX, 16.7% ESBL P. aeruginosa: PIP > CL > IMP	[40]
Ethiopia	2018	414, adult, CAP	Sputum	49.7%	K. pneumoniae (18.0%) and P. aeruginosa (11.4%)	K. pneumoniae: AMP/TE > AMC > SXT > C/DO > CN > CIP, 100% P. aeruginosa: CN > CRO > CIP > PIP > CAZ, 42.1%	[4]
Ethiopia	2020	406, ≥ 5 years, CAP	Sputum	56.7%	K. pneumoniae (28.0%) and P. aeruginosa (14.0%)	K. pneumoniae: AMP > TE > SXT/AMC > C > AK > CAZ > PZP/CXM > CIP, 97.7% P. aeruginosa: CAZ > CN > TE/PEP/AK > CIP, 45.5%	[37]
Ethiopia	2021	312, adult, CAP	Sputum	53.2%	K. pneumoniae (31.0%) and E. coli (20.7%)	K. pneumoniae: AMP > AMC > SXT > TE/DO, 94.9% E. coli: AMP > TE > DO > SXT > AMC, 93.8%	[38]

CAP community-acquired pneumonia, HAP hospital-acquired pneumonia, VAP ventilator-associated pneumonia, ESBL extended-spectrum beta-lactamase, AMP ampicillin, AMC amoxicillin/clavulanate, TOB tobramycin, TZP piperacillin/tazobactam, CRO ceftriaxone, AK amikacin, CXM cefuroxime, CTX cefotaxime, CIP ciprofloxacin, SXT trimethoprim-sulfamethoxazole, CL colistin, CN gentamicin, IMP imipenem, CAZ ceftazidime, TE tetracycline, C chloramphenicol, PEP cefepime, MER meropenem, MIN minocycline, APS ampicillin/sulbactam, TLV ticarcillin/clavulanate, LEV levofloxacin, AZT aztreonam, DO doxycycline

Table 2

Summary of the isolation and drug resistance profile of GNB in HAP/VAP

Country	Study year	Participants, age group, pneumonia category	Specimen	GNB (%) in culture	Frequently isolated GNB (%)	Decreasing order of resistance, MDR (%)	References
India	2012 to 2014	87, all age, VAP	Tracheal aspirates	88.3%	P. aeruginosa (38.3%), A. baumannii (15.6%), and K. pneumoniae (14.3%)	P. aeruginosa: PEP > CAZ > CN, 73.1% A. baumannii: CXT > CTX > PEP, 83.3% K. pneumoniae: CXT > CTX > PEP, 72.2%	[53]
Egypt	2014 to 2015	153, all age, VAP	Tracheal aspirates	87.1%	K. pneumoniae (36.9%), E. coli (21.04%), A. baumannii (14.95%), and P. aeruginosa (14.16%)	K. pneumoniae: CRO > PEP > AZT > CIP/TE > SXT > TZP > CN > AK > IMP, 100% E. coli: CTR > AZT/PEP > TE > CIP > SXT > CN > TZP > AK > IMP, 98% A. baumannii: CAZ/CTR/PEP/IMP/LEV/MER/PIP > AK > SXT > CN, 100% P. aeruginosa: CAR/CRO > CAZ > SXT/PIP/CIP/CN > AK/C > IMP/TZP, 84.84%	[54]
Bangladesh	2015 to 2016	51, all age, VAP	Tracheal aspirates	76.13%	A. baumannii (37.5%), K. pneumoniae (22.7%) and P. aeruginosa (13.6%)	A. baumannii: CRO/CAZ/CTX > AZT/PEP > AK/CN/COT > CIP > IMP > PZP K. pneumoniae: CRO/CAZ/CTX/PEP > CPR > SXT > CN > AK P. aeruginosa: PEP/CTX/CRO > CN/SXT/IMP > CAZ/CIP	[55]
Vietnam	2017 to 2018	103, 32 to 94 years old, HAP	Sputum and bronchoalveolar lavage	95.3%	A. baumannii (47.5%), K. pneumoniae (16.2%) and P. aeruginosa (12.1%)	A. baumannii: APS/PEP/CTX/CAZ/CRO/MER/CIP/LEV > PZP > TLV > TOB, 100% K. pneumoniae: AMP > TOB/PEP/CAZ/SXT > TLV/CRO > AK/CN, 72.7% P. aeruginosa: CTX > CRO/TLV/CN > TOB/PEP/CIP > AK, 92.3%	[56]
Tanzania	2019 to 2020	269, adult, VAP	Bronchial aspirate	80.1%	P. aeruginosa (24.7%),K. pneumoniae (19.8%), and E. coli (12.4%)	P. aeruginosa: CAZ/CXM/AZT/CIP > CN, 73.9% K. pneumoniae: CRO > AMC > CIP > PIP, 76.4% E. coli: CN > CRO > CAZ, 70%	[57]

CAP community-acquired pneumonia, HAP hospital-acquired pneumonia, VAP ventilator-associated pneumonia, ESBL extended-spectrum beta-lactamase, AMP ampicillin, AMC amoxicillin/clavulanate, TOB tobramycin, TZP piperacillin/tazobactam, CRO ceftriaxone, AK amikacin, CXM cefuroxime, CTX cefotaxime, CIP ciprofloxacin, SXT trimethoprim-sulfamethoxazole, CL colistin, CN gentamicin, IMP imipenem, CAZ ceftazidime, TE tetracycline, C chloramphenicol, PEP cefepime, MER meropenem, MIN minocycline, APS ampicillin/sulbactam, TLV ticarcillin/clavulanate, LEV levofloxacin, AZT aztreonam, DO doxycycline

Drug resistance patterns of GNB

Drug resistance in GNB varied from place to place and studies reported high drug resistance in elderly patients and all age groups in VAP. This may be due to the increased exposure to antibiotics in the elderly and the high frequency of MDR-GNB in the hospital areas related to VAP (Tables 1 and 2). In a study of drug resistance analysis on older CAP outpatient reports, ESBL producing strains were detected in E. coli (42.9%) and K. pneumoniae (25.0%) [26]. A study in Ethiopian adult CAP patients showed MDR prevalence of 100% in K. pneumoniae, P. vulgaris, and H. influenzae, 90% in E. coli, and 83.3% in P. mirabilis isolates [4]. Antimicrobial resistance in GNB responsible for 45–70% of VAP, is a daily challenge to ICU physicians [59]. An epidemiological study on VAP patients reported 72.1% of MDR-GNB [53].

Why has pneumonia etiology shifted to GNB?

Most of the people in the community, particularly in low-income countries, purchase cheap and freely available antibiotics from local drug stores and use them without a physician’s prescription, resulting in the ineffective killing of the causative agent, treatment failure, and the survival of resistant GNB, which increases the percentage of GNB resistant to drugs [60]. Poor infection control, inadequate antimicrobial stewardship, the limited vaccine coverage targeting GNB, their high burden in the hospital settings as a source of drug-resistant GNB spread to the community through hospital effluents, the difficult nature of acquiring resistance through transmissible genes, which act as a vector or reservoir of resistant genes, the increased comorbid conditions, the increased elderly populations, and the aggressive virulence determinants to cause severe disease are all reasons for the colonization of GNB [13, 61, 62].

Risk factors for MDR-GNB

According to the studies report [63-65], MDR-GNB causing pneumonia can be acquired from the community or hospital setting with risk factors including prolonged hospital stay, prior MDR-GNB colonization or infection, high frequency of antibiotic resistance in the setting, ICU admission, mechanical ventilation, and surgical intervention. They are also common in the elderly population, patients with prior antibiotic use, those with underlying pulmonary diseases (such as chronic obstructive pulmonary disease and bronchiectasis), diabetes mellitus, immunosuppressive conditions (like HIV and malignancies), prior hospitalization, and chronic alcoholism [66-70]. In addition, enteral malnutrition and the use of carbapenem drugs are significant risk factors for PDR A. baumannii-induced VAP [71].

Pathogenesis: the role of virulence determinants in transmission, colonization, adhesion, and invasion

Gram-negative bacterial pneumonia can be acquired through the aspiration of bacteria from parts of the upper respiratory tract or gastrointestinal tract (GIT), the inhalation of aerosols, hematogenous spread from distant sites such as the urinary tract, GIT, or lungs infected with E. coli and P. aeruginosa into the alveoli [72]. Additionally, bacterial translocation from the GIT has recently been known to be a mechanism of acquiring pneumonia [73]. Among these routes, aspiration is a common cause of HAP and CAP [74]. Approximately 45% of healthy adults aspirate oropharyngeal bacteria while sleeping, and abnormal swallowing of bacteria may also occur in people with depressed consciousness, respiratory tract instrumentation, and/or mechanically ventilated patients [75].

Bacterial translocation is a pathogenesis mechanism in which viable bacterial flora or enteric microorganisms of the GIT escape from the intestinal lumen through epithelial mucosa into the mesenteric lymph nodes and then, possibly, to the lung. The translocating organisms could be the cause of pneumonia, or they could cause changes in defense mechanisms that make it difficult for the host to clear a bacterial inoculum from the lungs. Patients with immunosuppression, cancer, or burns may experience this [76, 77].

The colonization of GNB substantially increases with previous use of antimicrobial agents in patients who have alcoholism, diabetes mellitus, pulmonary disease, or use of inhalation devices [78]. Adhesins, invasins, secretory molecules such as effectors and extracellular matrix, outer membrane vesicles, toxins, capsules, fimbriae, flagella, iron acquisition systems consisting of an outer membrane receptor, a periplasmic binding protein, and an inner membrane ABC transporter, and biofilm formation in GNB contribute to disease occurrence. Some of these processes, like adhesins, are found in chromosomes, whereas others, such as plasmids, are found in mobile genetic components. Siderophores, for example, are virulence factors that allow bacteria to adapt and live in a host by competing with normal flora for iron [79].

Adherence is aided by adhesions, which are bacterial surface structures that promote attachment to epithelial cells, pili, cilia, capsules, elastase production, host factors like surface proteins and polysaccharides, and environmental factors like pH and the presence of mucin in respiratory secretions. Malnutrition, severe illness, endotracheal intubation, and the postoperative state can all increase GNB adherence. Prolonged intubation causes the biofilm formation on the inner surface of the endotracheal tube, which contributes to pathogen persistence and treatment failure [80]. Invasion of GNB into the spaces between cells and adjacent alveoli via flagellar movement across the connecting pores causes neutrophil recruitment and cytokine release, resulting in immune system activation and inflammatory response. Due to lipopolysaccharide endotoxin, this inflammatory response is the primary cause of general respiratory symptoms such as fever, chills, fatigue, changes in blood pressure, and even shock. Neutrophils, bacteria, and fluid leaking from nearby blood vessels fill the alveoli, causing dyspnea due to impaired oxygen transportation. Severe pneumonia causes hypoxia, which leads to hyperventilation and death [81, 82].

Challenges and major advancements in the diagnosis of pneumonia

Accurate pneumonia diagnosis is critical for determining the disease burden and developing effective treatment and prevention strategies. Currently, pneumonia is diagnosed based on the patient’s medical history and clinical signs and symptoms like cough, fever, purulent sputum, auscultation findings, acute pulmonary infiltrate, and dyspnea. Chest radiography and computed tomography scans are common radiographic imaging techniques used in the diagnosis of pneumonia [83]. It is impossible to determine the etiology of pneumonia based solely on clinical examination; instead, an optimal specimen must be obtained for laboratory identification of bacteria [84]. The inability to obtain good quality sputum due to contamination with normal respiratory flora, the good safety profile of transthoracic lung aspirates, and the difficulty of obtaining sputum in children and the elderly all posed challenges [85]. Routine culture, bacterial identification, and antimicrobial susceptibility testing need different specimens, require specimen treatment, poor detection rate, and long turnaround times up to 48–72 h.

Molecular diagnostic tests and/or nucleic acid detection tests have been used for the diagnosis of bacterial pneumonia over recent years. Rapid molecular detection of the pathogen can minimize the empirical use of broad-spectrum antibiotics in severe CAP, HAP, and VAP, but their interpretation is difficult due to differences in the local treatment guidelines and resistance genes, the discrepancy between genotype and phenotype, the ongoing discovery of new resistance mechanisms, and, as a result, the potential presence of unknown mechanisms, which may lead to false-negative results using molecular techniques [86]. A study by Kitsios et al. [87] about the etiologic diagnosis of bacterial pneumonia in mechanically ventilated patients reported that enhanced pathogen detection using microbial DNA sequencing can improve upon culture-based diagnosis, that sequencing profiles correlate with the host response, and offers substantial opportunity for individualized therapeutic targeting and antimicrobial stewardship. The multiplex polymerase chain reaction (M-PCR) has become useful for the rapid diagnosis of bacterial causes of pneumonia directly from the sputum and blood [88]. A recent prospective study used M-PCR to detect bacterial pathogens in 95 clinical bronchoalveolar lavages or plugged telescoping catheter samples from VAP patients and found that the M-PCR system had a global sensitivity of 80% and specificity of 99%. The sensitivity was better for GNB identification (90%) [89]. The Bio Fire Film Array Pneumonia Plus Panel is an FDA-cleared sample-to-answer assay that enables the detection of bacteria and antimicrobial resistance marker genes from sputum and bronchoalveolar lavage fluid [90]. It has a shorter turn-around-time than culture-based approaches, is more beneficial to a diverse set of patients with severe LRTI, such as severe CAP or VAP, who are routinely prescribed broad-spectrum empirical treatment, and can change antibiotic prescriptions in 40.7% of patients [91].

Antimicrobial resistance mechanisms in GNB

Resistance to antimicrobial agents is increasing at both community and hospital levels, being especially relevant in the hospital settings in which changes in the hospital environment and strong selective pressure favor the selection, persistence, and maintenance of resistant, MDR (resistant to at least one agent in three or more antimicrobial classes), XDR (resistant to at least one agent in all but two or fewer antimicrobial classes), and even PDR strains (resistant to all the current groups of antibiotics for therapeutic use), causing antibiotic treatment failure, increased mortality, and morbidity, and having a significant impact on the cost of medical treatment and prevention of bacterial infections [92]. Antimicrobial resistance can be innate resistance by genes encoding inherent antibiotic resistance present in the bacteria, acquired resistance due to selective antibiotic pressure from the environment, or adaptative resistance that is a reflection of the ecological niche of the bacteria, including environmentally induced genetic changes. Mechanisms of antibiotic resistance include target alteration of the drug, the impermeability of the bacteria, bypassing the drug, efflux of the drug, biofilm formation, and genetically associated changes such as mutations and plasmid-mediated transfer of resistance genes [93, 94]. Additionally, GNB is resistant to antibiotics with an alteration in the outer membrane such as porin mutations, production of enzymes including beta-lactamase, carbapenemase, and aminoglycoside modifying enzymes (phosphorylating, adenylylating, and acetylating enzymes), and increased expression of the transmembrane efflux pump [95].

Alexander Fleming first discovered resistance in gram-negative species to beta-lactam antibiotics in 1929. Then now, resistance to beta-lactam drugs in GNB has frequently been studied and it is due to the production of beta-lactamase enzymes such as the active site serine beta-lactamases (classes A, C, and D) and the class B Metallo-beta-lactamases that use active site zinc ions to coordinate a nucleophilic hydroxide to mediate ring-opening [96]. The enzyme lactamase is formed in the periplasmic space, which inactivates the antibiotic after penetration into the bacterial organism and breaks the amide bond of the four-membered beta-lactam ring, deactivating the molecule’s antimicrobially active molecules through hydrolysis. The highly drug-resistant P. aeruginosa, A. baumannii, and K. pneumoniae in HAP and VAP patients encodes plasmid-mediated AmpC b-lactamases on their chromosomes that hydrolyze cephalosporins, monobactams, and cephamycins, as well as the expression of class A KPC b-lactamases, confer resistance to carbapenems [97]. Moreover, the loss of OprD associated with resistance to carbapenems such as imipenem and meropenem in P. aeruginosa and increased production of drug efflux pump systems (mex), as part of either an acquired or intrinsic resistance repertoire, is capable of exporting various substrates from the periplasm of GNB to the surrounding environment before the action of the drug [98].

Biofilm-mediated resistance

A biofilm is an aggregate of microorganisms that are firmly attached to the biotic or abiotic surface, encased within an extracellular polymeric substance matrix, and that can show new characteristics to gene expression, protein synthesis, growth rate, and metabolic activities, thereby facilitating the anchorage to any surface irreversibly. The matrix confers antibiotic resistance through processes such as slow penetration of antibiotics, expression of chromosomally encoded resistant genes or development of persistent cells, changes in bacterial growth rate and metabolic activities, altered microenvironment due to depletion of nutrients and/or accumulation of waste substances that will antagonize the action of antibiotics, and even counteracting the host immunity [99]. In mechanically ventilated patients, biofilms are associated with endotracheal intubation, which acts as a reservoir for drug-resistant pathogens causing VAP such as P. aeruginosa and A. baumannii that persist in the hospital settings [100].

Update on treatment options and promising future perspectives

Antibiotics are the treatment of choice for bacterial pneumonia, and the choice of antibiotic depends on the nature of pneumonia, the microorganism, and the immune status of the individual. In randomized double-blind trials, omadacycline, lefamulin, and delafloxacin were non-inferior to moxifloxacin for treating community-acquired bacterial pneumonia in adults [101-103]. The ongoing spread of antimicrobial resistance in pneumonia cases has made treating MDR-GNB empirically difficult [104]. A review by James et al. [23] about the novel antibiotics for the treatment of HAP and VAP caused by resistant GNB reported that ceftazidime has demonstrated noninferiority to meropenem against carbapenem-resistant Enterobacteriaceae (CRE) and ceftolozane against MDR P. aeruginosa. Recent noninferiority trials reported cefiderocol, ceftolozane/tazobactam, ceftazidime/avibactam, and imipenem/cilastatin/relebactam combinations as potential options in patients with MDR gram-negative nosocomial pneumonia, which showed non-inferior to high-dose, extended-infusion carbapenems such as meropenem in terms of clinical efficacy and all-cause mortality [105-108].

Aerosolized antibiotic therapy is already widely administered in ICUs during mechanical ventilation. A single-center, double-blind study on adjunctive therapy of ICU patients with confirmed MDR-GNB in VAP reported that aerosolized amikacin successfully eradicated existing MDR bacteria without inducing new resistance to amikacin or change in serum creatinine [109]. Colistin is a last resort therapy for infections caused by MDR-GNB, in particular P. aeruginosa, A. baumannii, and K. pneumoniae [110]. In critically ill patients with nosocomial pneumonia caused by MDR-GNB, including carbapenem-resistant strains, adjunctive nebulized colistin therapy provided non-inferior therapeutic efficacy to parenteral colistin therapy with lower clinical failure [111, 112].

The therapeutic potential of bacteriophages targeting MDR strains of GNB using animal models was evaluated in vitro and showed high infectivity of phages and multiple phage doses were required for effective treatment in vivo [113]. Novel treatment options such as PlyF307 lysine phage against MDR A. baumannii, VTCCBPA43 phage against virulent K. pneumoniae, PlyPa91 and vB_PaeP_PA01EW phages against P. aeruginosa, and Abp95 lytic myoviridae phage against multi-genotypes of carbapenem-resistant A. baumannii were demonstrated in mouse models [114-118]. Tridecaptins, a non-ribosomal lipopeptide, showed a selective bactericidal activity against the gram-negative version of the peptidoglycan precursor lipid II on the outer leaflet of the inner membrane and disrupts the proton-motive force [119].

In a mouse infection model, odilorhabdins also showed antimicrobial activity against GNB, including CRE, by binding to the small ribosomal subunit at a site not exploited by common antibiotics that induce miscoding, amino acid misincorporation, and bypass premature stop codons that interfere with protein synthesis [120]. Quorum sensing inhibitors can be applied along with other antibiotics such as Isobutyl-4, 5-Dihydroxy-2, 3-pentanedione (DPD) and phenyl-DPD with gentamicin and small molecules to fight biofilm-mediated drug resistance [60]. A recent study found that maipomycin A, a novel natural compound with promising anti-biofilm activity against pathogenic GNB, acts as a synergist to enhance colistin efficacy against A. baumannii [121]. In situations where antimicrobial treatment has been unsuccessful or where current therapies have caused resistance, iron-chelation therapy reduces the growth of MDR-GNB and potentiates antimicrobial strategies, particularly in HAP/VAP [122].

GNB vaccine trials are currently underway

Even if vaccines against pneumonia were introduced in routine immunization programs, reaching for all people in low-income countries is a common challenge to tackling pneumonia. Although there is no licensed vaccine for clinical use against GNB, there is a new advance in vaccination. The polysaccharide capsules of K. pneumoniae have been previously targeted for developing therapeutics and vaccines for treating carbapenem-resistant K. pneumoniae infections [123]. A recent trial in a mouse model demonstrated the promising efficacy of new vaccine containing YidR recombinant protein to prevent K. pneumoniae disease [124]. The preclinical study reported that K. pneumoniae bioconjugates are immunogenic and effective, protecting mice against lethal infection from 2 hvKp strains, NTUH K-2044 and ATCC 43816 [125].

Kumar et al. [126] evaluated the potential of recombinant FyuA of K. pneumoniae against lung infection in BALB/c mice and found that immunization generated both humoral and cell-mediated responses that conferred protection against the lethal dose of bacteria. A randomized clinical trial evaluated recombinant IC43 100 μg vaccination against potentially lethal P. aeruginosa infection in mechanically ventilated non-surgical ICU patients and found that it was both immunogenic and well-tolerated [127]. A live vaccine containing auxotrophic strain that lacks the key enzyme involved in D-glutamate biosynthesis, a structural component of the bacterial cell wall, confers mucosal immunity and protection against lethal pneumonia caused by P. aeruginosa [128].

Role of Stenotrophomonas maltophilia in pneumonia: an opportunistic GNB

S. maltophilia is an aerobic, non-fermenting, and environmental MDR-GNB, emerges in immunocompromised individuals and causes severe pneumonia [129]. It causes HAP in critically ill patients in the ICU due to its ability of biofilm formation and site adhesion in respiratory instruments and its intrinsic and acquired resistance to various antibiotics makes treatment difficult [130, 131]. In recent studies, the incidence of VAP due to S. maltophilia was 0.27 to 0.93% [131-133]. It causes severe hemorrhagic pneumonia with a reported mortality rate of 100% [134]. A study reported high mortality of S. maltophilia pneumonia in older cancer patients who used inappropriate antibiotic treatment [135]. Hemorrhagic pneumonia caused by S. maltophilia is a significant risk factor for mortality in patients with hematologic malignancy such as thrombocytopenia and prolonged neutropenia [136]. Currently, tigecycline is a promising alternative to trimethoprim-sulfamethoxazole and fluoroquinolones for treating VAP caused by S. maltophilia, but its resistance to available antibiotics has increased [137].

Conclusion and recommendations

Worldwide, the prevalence of GNB among pneumonia patients is in the range of 49.7% to 95.3%. The predominant MDR-GNB in recently published studies causing pneumonia were A. baumannii, K. pneumoniae, and P. aeruginosa, with A. baumannii isolated particularly in VAP patients. The prevalence of MDR-GNB is higher in the elderly population, prior MDR-GNB infection, prolonged hospital stays, ICU admission, mechanical ventilation, surgical intervention, prior antibiotic use, comorbidity, chronic alcoholism, and enteral malnutrition. Although the resistance pattern of GNB varies from place to place, their resistance to commonly used antibiotics is almost similar in all studies across the country. In the majority reports of GNB, ampicillin, tetracyclines, and amoxicillin-clavulanic acid were highly resistant in CAP, whereas cephalosporins and carbapenems were in VAP. S. maltophilia became a severe cause of HAP in critically ill patients due to its ability of biofilm formation, site adhesion in respiratory equipment, and its intrinsic and acquired drug resistance mechanism. Microbial DNA sequencing, M-PCR, and the Bio Fire Film Array Pneumonia Plus Panel have been recently applied to detect bacterial pneumonia. Novel PCR-based techniques should be implemented for the early detection of drug-resistant genes to overcome the transmission of highly resistant genes between bacteria. Since there are increased MDR and PDR gram-negative strains, it makes the treatment more complicated, which may lead to high morbidity, economic losses, and mortality. To this end, newer, effective combination therapies with minimal clinical side effects, antibiotics against drug-resistant genes, antibiofilm agents, and vaccine approaches involving genetic vaccines or pathogen-specific lymphocytes, particularly for PDR strains, should be developed.