Contents

1

.Multidrug-resistant Bacteria (MDR) (Ruihao Zhang)................................................1

1

.1.Multidrug-resistant Bacteria Definition............................................................1

.2 Multidrug-resistant Bacteria Monitoring, Reporting, Disposal ........................1

.3 Focused Surveillance of MDRO Species..........................................................2

1

.3.1 Introduction.............................................................................................2

.3.2 Carbapenem-resistant Enterobacteriaceae (CRE)...................................3

.3.3 Methicillin-resistant Staphylococcus Aureus (MRSA)...........................3

.3.4 Vancomycin-resistant Enterococcus (VRE)............................................3

.3.5 Carbapenem-resistant Acinetobacter Baumannii (CRAB) .....................3

.3.6 Carbapenem-resistant Pseudomonas aeruginosa (CRPAE) ....................3

.3.7 Conclusion ..............................................................................................3

1

.4 the Risk Factors for MDR Infection .................................................................4

.5 MDRO Transmission Routes and Infection Hazards........................................5

1

.5.1 Transmission Routes...............................................................................5

.5.2 Infection Hazards....................................................................................5

2

3

4

.The mechanism about multidrug resistant bacteria(Haotian Sun)..............................6

2

.1Mechanisms of action of antibacterial agents....................................................6

.2 Mechanisms of multidrug-resistant bacteria drug resistance............................7

2

.2.1 Limiting uptake of drugs.........................................................................8

.2.2 Modification of drug targets ...................................................................8

.2.3 Drug inactivation ....................................................................................8

.2.4 Drug efflux..............................................................................................8

.2.5 Case analyzing of β-Lactam resistance in Gram-positive and Gram-

negative bacteria ..............................................................................................9

2

.3 Mechanisms of immune response of bacteria infection..................................11

. Strategies for immune escape of multi-drug resistant bacteria(Manda Sun)...........14

.1 Multidrug-resistant bacteria can evade immune system attacks through many

strategies ...............................................................................................................14

3

.2 Suppression of Phagolysosome Maturation....................................................15

.3 Escape from Oxidative Killing by Neutrophils ..............................................16

.4 Neutralization of antimicrobial peptides (AMPs) in multidrug-resistant

bacteria..................................................................................................................16

3

.5 Manipulating Host Cell Responses for Immune Evasion...............................17

.6 Interplay with Antigen Presentation Dynamics and the Role of Antigen Export

.7 Escape from the phagosome before the fusion begins....................................19

.Approaches for the treatment of infections due to multidrug-resistant bacterial

pathogens(Wanting Hao) .............................................................................................21

4

.1 Antimicrobial peptides-based therapies..........................................................22

.2 Phage-based therapies.....................................................................................25

.3 Immunomodulatory therapy............................................................................25

.4 Antibody-mediated therapy.............................................................................26

.5 Macrophage cell therapy.................................................................................27

.6 Targeting unconventional targets ....................................................................27

.7 Allosteric target...............................................................................................27

.8 Conclusion ......................................................................................................28

1

.Multidrug-resistant Bacteria (MDR)

1

.1. Multidrug-resistant Bacteria Definition

Multidrug-resistant organisms (MDROs), primarily bacteria, pose a significant

[1]

challenge in the field of healthcare. These organisms exhibit resistance to three or

more classes of antimicrobial drugs used in clinical practice simultaneously. The drugs,

structurally different and with varying mechanisms of action, are no longer effective

against these resistant strains.

The phenomenon of multidrug resistance includes extensive drug resistance (XDR)

and pan-drug resistance (PDR). XDR bacteria are insensitive to all antimicrobials

except those in groups 1-2 (mucins or cyclins). On the other hand, PDR bacteria are

insensitive to all drugs across all antimicrobial classes.

Figure 1.1 Relationship Diagram of Multidrug-resistant Bacteria Definition

The emergence of bacterial resistance is a natural phenomenon resulting from the

evolutionary selection of bacteria. However, this process has been exacerbated by the

misuse of antibiotics. The overuse and inappropriate use of antibiotics have accelerated

the development of resistance, making previously effective treatments obsolete.

Understanding the mechanisms behind multidrug resistance is crucial for

developing new therapeutic strategies and mitigating the impact of these superbugs on

public health.

1

.2 Multidrug-resistant Bacteria Monitoring, Disposal

Multidrug-resistant bacteria are organisms that have developed resistance to

multiple types of antibiotics. These bacteria pose a significant challenge to public health

due to the difficulty in treating infections caused by them. The monitoring, reporting,

and disposal of these bacteria are crucial in controlling their spread and developing

[2]

effective treatment strategies.

1

The first step in managing multidrug-resistant bacteria is identifying patients with

infectious diseases. Once these patients are identified, their pathogenicity is sent for

testing. Laboratory microbiology plays a vital role in this process, where bacterial

culture is performed to identify the presence of bacteria and their resistance profile.

The outcomes of bacterial culture can be broadly classified into two categories:

bacteria-positive and bacteria-negative. In bacteria-positive cases, the bacteria are

further classified into multidrug-resistant and non-multidrug-resistant. Each outcome

has different implications for patient management and treatment strategies.

For multidrug-resistant cases, the Hospital Infection Management Section

provides guidance, supervision, and inspection. The laboratory department issues and

labels a test report, which is then sent to the clinical department. The clinical department

plays a crucial role in managing these cases and deciding the appropriate treatment plan.

For multidrug-resistant cases, contact quarantine is implemented until the clinical

symptoms have improved or the patient is cured. This step is crucial in preventing the

spread of multidrug-resistant bacteria to other patients and healthcare workers.

The effective monitoring, reporting, and disposal of multidrug-resistant bacteria

are crucial in managing the spread of these organisms and ensuring effective treatment

strategies. Future research should focus on understanding the mechanisms of resistance

in these bacteria and developing new antibiotics that can effectively treat infections

caused by them.

Figure 1.2 Multidrug-resistant Bacteria Monitoring, Reporting, Disposal Flowchart

1

.3 Focused Surveillance of MDRO Species

.3.1 Introduction

2

The rise of multidrug-resistant organisms (MDROs) poses a significant threat to

global health. This paper focuses on five key MDRO species, as identified in the

provided image: Carbapenem-resistant Enterobacteriaceae (CRE), Methicillin-resistant

Staphylococcus Aureus (MRSA), Vancomycin-resistant Enterococcus (VRE),

Carbapenem-resistant Acinetobacter Baumannii (CRAB), and Carbapenem-resistant

Pseudomonas aeruginosa (CRPAE).

1

.3.2 Carbapenem-resistant Enterobacteriaceae (CRE)

CRE, including species such as Escherichia coli and Klebsiella pneumoniae, pose

a significant threat due to their high level of resistance to carbapenems, a class of

[3]

antibiotics that are often used as a last resort for treating severe infections. The

prevalence of CRE in healthcare settings is a major concern, making the surveillance

of these organisms crucial for controlling their spread and developing effective

treatment strategies.

1

.3.3 Methicillin-resistant Staphylococcus Aureus (MRSA)

MRSA is a well-known MDRO that has shown resistance to many antibiotics,

[4]

including methicillin, a type of penicillin. MRSA is commonly found in hospital

settings, where it can cause severe infections such as pneumonia and bloodstream

infections if not properly managed.

1

.3.4 Vancomycin-resistant Enterococcus (VRE)

VRE, specifically Enterococcus faecium and Enterococcus faecalis, are of

particular concern due to their resistance to vancomycin, a last-resort antibiotic for

[5]

many gram-positive infections. The emergence of VRE has been associated with

increased morbidity and mortality, particularly among immunocompromised patients.

1

.3.5 Carbapenem-resistant Acinetobacter Baumannii

(CRAB)

CRAB is a growing concern in healthcare settings, particularly in intensive care

units.^[⁶^]CRAB is known for its ability to survive in the environment for extended

periods, contributing to its spread. Its resistance to carbapenems, a class of broad-

spectrum antibiotics, makes it a challenging organism to treat.

1

.3.6 Carbapenem-resistant Pseudomonas aeruginosa

(CRPAE)

3

CRPAE is a common pathogen in healthcare settings, known for its intrinsic

[7]

resistance to many antibiotics and disinfectants. CRPAE can cause a range of

infections, including pneumonia, urinary tract infections, and bloodstream infections,

particularly in patients with weakened immune systems.

1

.3.7 Conclusion

The focused surveillance of these MDRO species is vital for early detection,

prevention of spread, and effective treatment planning. Continued research and

surveillance efforts are needed to combat the growing threat of MDROs. This includes

the development of new antibiotics, the implementation of robust infection control

measures, and the promotion of antibiotic stewardship to prevent the further emergence

and spread of resistance.

1

.4 the Risk Factors for MDR Infection

Elderly Population: The elderly population is more susceptible to MDR

infections due to several factors. As individuals age, there is a natural decline in

physiological and metabolic functions, and the immune system becomes less effective.

This lowered immune function, combined with the increased likelihood of chronic

conditions and hospitalizations, makes the elderly population particularly vulnerable to

MDR infections.

Immunocompromised Individuals: Patients with conditions that compromise

their immune system, such as diabetes mellitus, chronic obstructive pulmonary disease,

cirrhosis of the liver, and uremia, are at a higher risk of MDR infections. Additionally,

individuals who have been treated with immunosuppressants for an extended period or

those receiving radiotherapy or chemotherapy also fall into this high-risk category.

These conditions and treatments can weaken the immune system, making it harder for

the body to fight off infections.

Invasive Operations: Invasive operations can destroy the barrier of the skin and

mucous membranes, which are the body’s first line of defense against pathogens. This

damage can lead to both exogenous infections (caused by pathogens from the

environment or other individuals) and endogenous infections (caused by bacterial

translocation within the body).

Antibiotic Treatments: Recent treatment with three or more antimicrobial drugs

within 90 days significantly increases the risk of MDR infection. This is because the

use of these drugs can kill off the non-resistant bacteria, allowing the resistant bacteria

to thrive and multiply.

Previous Hospitalisation: Longer hospital stays increase the risk of hospital-

acquired infections, also known as nosocomial infections. These environments often

4

have a higher prevalence of MDR organisms, increasing the chance of patients

acquiring these infections.

Past Medical History: A previous history of MDR colonization or infection

increases susceptibility to future MDR infections. This is because once a person has

been colonized or infected with an MDR organism, they may become a carrier of that

organism, increasing the risk of future infections.

Understanding these risk factors is crucial for the prevention and management of

MDR infections. ^[⁸^-¹⁰^]Further research is needed to develop effective strategies to

mitigate these risks, such as improved infection control practices, antibiotic stewardship

programs, and the development of new antimicrobial agents. It’s also important to

educate healthcare providers and patients about these risk factors and the importance of

preventative measures.

1

.5 MDRO Transmission Routes and Infection Hazards

.5.1 Transmission Routes

Contact Transmission: Contact transmission is the most common mode of

transmission for MDROs. This can occur either directly, through person-to-person

contact, or indirectly, through contact with contaminated surfaces. For example,

healthcare workers can inadvertently transmit MDROs to patients through their hands

[11]

or medical equipment.

Droplet Transmission: Droplet transmission occurs when bacteria are spread

through small droplets that are expelled during operations like coughing, sneezing, or

[12]

suctioning. Aerosol-forming procedures, such as intubation or bronchoscopy, can

also lead to droplet transmission. These droplets can land on surfaces or be inhaled by

people nearby, leading to potential infection.

Airborne Transmission: Airborne transmission can occur when MDROs

[13]

contaminate air conditioning vents or other air circulating systems. This can lead to

the widespread dissemination of these organisms within a facility. Regular maintenance

and cleaning of these systems are crucial preventative measures.

1

.5.2 Infection Hazards

Higher Morbidity and Mortality: Patients infected with MDROs face higher

morbidity and mortality rates than those infected with sensitive organisms. This is due

to the difficulty in treating these infections and the potential for complications.

Limited Antimicrobial Choices: The clinical presentation of infections with

[14]

MDROs is often consistent with those of sensitive strains of bacteria. However, the

5

choice of antimicrobials for treatment is extremely limited due to the resistance of these

organisms to multiple drugs.

Prolonged Hospital Stays and Increased Costs: MDRO infections can lead to

prolonged hospital stays due to the need for extended treatment and isolation measures.

This can result in increased costs for diagnosis, treatment, and additional care.

Increased Risk of Adverse Reactions: The limited choice of antimicrobials for

treating MDRO infections can lead to the use of less commonly used drugs, which may

have a higher risk of adverse reactions.

Source of Communication: MDROs can become a source of communication in

[15]

the sense that their presence can indicate issues with infection control practices.

Monitoring and reporting of MDRO infections are important for identifying and

addressing these issues.

6

2.The mechanism about multidrug resistant

bacteria

2

.1Mechanisms of action of antibacterial agents

The development of new antibiotics by the pharmaceutical industry in countries

like the United States, Japan, the United Kingdom, France, and Germany has created a

false sense of security among society and the scientific community regarding bacterial

resistance. With a wide range of antibiotics available, including various penicillins,

cephalosporins, tetracyclines, aminoglycosides, and others, it is easy to believe that we

[16-17]

are well-equipped to combat bacterial infections

. However, the reality is that

people still die from drug-resistant bacterial infections, even in major cities around the

world like New York, San Francisco, Paris, Barcelona, Tokyo, or Singapore.

[20]

Figure 2.1 Mechanisms of different antibacterial drugs acting on bacteria

Antimicrobial agents can become ineffective due to three main mechanisms.

Firstly, the antibiotic can be inactivated through destruction or modification. For

example, enzymes like β-lactamase and aminoglycoside-inactivating enzymes can

modify the antibiotic and render it ineffective.

7

Secondly, the antibiotic's access to its target can be prevented. This can occur

through changes in permeability or the efflux of certain agents. For instance, bacteria

can develop mechanisms to block the entry of antibiotics such as β-lactams,

aminoglycosides, and tetracyclines, or they can actively pump out the antibiotic to

avoid its effects.

Lastly, the target site of the antibiotic can be altered. Even a single amino acid

change in an enzyme can result in a modified target site that is no longer susceptible to

the antibiotic. This can happen with antibiotics like β-lactams, macrolides, and folate

synthesis antagonists.

These mechanisms of antibiotic resistance demonstrate the ability of bacteria to

evade the effects of antimicrobial agents, which poses a significant challenge in treating

[18-19]

bacterial infections

.

2

.2 Mechanisms of multidrug-resistant bacteria drug

resistance

Antimicrobial resistance mechanisms can be broadly classified into four main

groups: (1) preventing the entry of a drug; (2) modifying a drug's target; (3) inactivating

a drug; and (4) actively expelling a drug. Figure 2 offers a comprehensive overview of

[21- 22]

these general antimicrobial resistance mechanisms.

[22]

Figure 2.2 General antimicrobial resistance mechanisms.

2

.2.1 Limiting uptake of drugs

Gram-negative bacteria possess an outer membrane that acts as a barrier to drug

8

entry.^[²³^-²⁴^]By modifying the structure or composition of the outer membrane, bacteria

can decrease the permeability of drugs. This can be achieved by reducing the number

of porins, channels that allow the passage of drugs, or by modifying the porins to restrict

[23]

drug entry . Also, bacteria possess transporters that facilitate the uptake of essential

nutrients, as well as certain drugs. By downregulating the expression of these

transporters or acquiring mutations in their genes, bacteria can reduce the entry of

drugs^[

25]

.

2

.2.2 Modification of drug targets

Bacteria can develop drug resistance through the modification of drug targets, a

mechanism that alters the molecular targets of drugs, rendering them less effective.

Bacteria can modify the specific binding sites targeted by drugs, such as antibiotics.

By acquiring mutations in the genes encoding these targets, bacteria can change the

structure and composition of the binding sites. This modification reduces the affinity of

drugs for their targets, decreasing the effectiveness of the drugs in inhibiting bacterial

growth or killing the bacteria. Some bacteria produce enzymes that chemically modify

drug targets. For example, bacteria may produce enzymes that methylate or acetylate

specific sites on proteins targeted by antibiotics. These enzymatic modifications can

[26-27]

disrupt the binding of drugs to their targets, leading to decreased drug efficacy

.

2

.2.3 Drug inactivation

Bacteria have two main methods of rendering drugs ineffective: breaking down

the drug or modifying it with a chemical group. The breakdown of drugs, including

widely used β-lactam antibiotics, is primarily carried out by an extensive group of

enzymes known as β-lactamases through hydrolysis. For tetracycline, the tetX gene

[28]

facilitates drug inactivation through hydrolysis . Another common resistance

mechanism involves modifying drugs through the transfer of chemical groups, such as

[29]

acetyl, phosphoryl, or adenyl, using various transferase enzymes

.

2

.2.4 Drug efflux

Bacteria can produce efflux pumps, membrane proteins that actively pump drugs

out of the bacterial cell. These pumps recognize and expel a wide range of drugs,

including antibiotics. By continuously pumping out drugs, bacteria can maintain lower

intracellular drug concentrations, reducing their effectiveness. Efflux pumps can be

[30]

constitutively active or induced by exposure to drugs, leading to multidrug resistance.

9

[31]

Figure 2.3 General structure of main efflux pump families.

2

.2.5 Case analyzing of β-Lactam resistance in Gram-

positive and Gram-negative bacteria

β-Lactam antibiotics exert their bactericidal activity by covalently binding to

penicillin-binding proteins (PBPs) and inactivating them, leading to disruption of

bacterial peptidoglycan synthesis and restructuring. In Gram-positive bacteria,

resistance to β-lactams primarily arises from alterations in PBPs, while enzymatic

[32]

degradation plays a lesser role in resistance

.

[32]

Figure 2.4 β-Lactam resistance in Gram-positive bacteria

Gram-negative bacteria develop resistance to β-lactam antibiotics through three

10

primary mechanisms: alterations in penicillin-binding proteins, production of β-

lactamases, and limited accessibility of target PBPs. Unlike in Gram-positive bacteria,

PBPs in Gram-negative bacteria are found in the periplasmic space. For β-lactam

antibiotics to reach their target sites, they must cross the bacterial outer membrane. Any

modifications that restrict the entry (such as porin loss) or lead to expulsion (via efflux

pumps) of β-lactam antibiotics will result in resistance. This resistance mechanism is

specific to Gram-negative bacteria. In Gram-negative bacteria, the predominant

resistance mechanism is the production of β-lactamases. Importantly, the combined

action of these diverse mechanisms collectively determines the ultimate expression of

[32]

resistance in Gram-negative bacteria.

[32]

Figure 2.5 β-Lactam resistance in Gram-negative bacteria

2

.3 Mechanisms of immune response of bacteria infection

When the human body is infected by bacteria, the innate immune system is the

first line of defense. The innate immune response is non-specific and acts quickly to

prevent the spread of the infection. The innate immune system firstly recognizes the

presence of bacteria through pattern recognition receptors (PRRs) such as Toll-like

receptors (TLRs) and NOD-like receptors (NLRs). These receptors can detect specific

molecular patterns on the surface of bacteria, known as pathogen-associated molecular

patterns (PAMPs). Upon recognition of PAMPs, immune cells such as macrophages,

neutrophils, and dendritic cells are activated. These cells play a crucial role in

11

phagocytosis, the process of engulfing and destroying bacteria. The activated immune

cells release signaling molecules called cytokines, such as interleukins and tumor

necrosis factor (TNF), triggering an inflammatory response. Inflammation helps to

recruit more immune cells to the site of infection and creates an unfavorable

environment for the bacteria. The complement system, a group of proteins in the blood,

is activated in response to bacterial infection. It helps in opsonization (marking bacteria

for phagocytosis), lysis of bacterial cells, and promoting inflammation. Chemokines, a

type of cytokine, are released to attract more immune cells to the site of infection,

amplifying the immune response. The innate immune system also activates

antimicrobial defenses, such as the production of antimicrobial peptides and enzymes,

to directly kill bacteria. Once the bacteria are cleared, anti-inflammatory signals help

[33]

in resolving the inflammatory response and tissue repair.

The innate immune response to bacterial infection is characterized by rapid

recognition and activation of immune cells, inflammation, and the initiation of

antimicrobial mechanisms to eliminate the invading bacteria. This initial response is

essential for containing the infection before the adaptive immune system, with its

specific immune response, can be fully activated.

[35]

Figure 2.6 Innate response of bacterial infection

After the innate immune response, the adaptive immune system is activated to

provide a specific immune response against the bacteria. Immune cells, such as

dendritic cells, phagocytose bacteria and process their antigens. These antigens are then

presented on the surface of the immune cells in complex with major histocompatibility

complex (MHC) molecules. Antigen-presenting cells present the bacterial antigens to

T cells, specifically CD4+ T helper cells and CD8+ cytotoxic T cells. This interaction

triggers activation and proliferation of these T cell subsets. The antigen-presenting cells

also interact with naïve B cells that have receptors specific to the bacterial antigens.

This interaction, along with cytokine signals from the activated T cells, leads to the

activation and differentiation of B cells into plasma cells. Plasma cells secrete

12

antibodies, also known as immunoglobulins (Ig), that are specific to the bacterial

antigens. Antibodies can directly neutralize bacteria, enhance phagocytosis by coating

bacteria (opsonization), or activate the complement system to promote bacterial lysis.

During the adaptive immune response, both T and B cells differentiate into memory

cells^[

34-35]

.

Memory cells have a long lifespan and provide rapid and enhanced response upon

re-exposure to the same bacterial antigen in the future. CD4+ T helper cells aid in the

cellular immune response by secreting cytokines that activate macrophages and

enhance phagocytosis. CD8+ cytotoxic T cells directly recognize and eliminate infected

cells harboring intracellular bacteria. The presence of memory cells allows for a swifter

and stronger immune response upon re-infection by the same bacteria. This is the basis

of immunity to certain bacterial infections.

The adaptive immune response to bacterial infection is a highly specific and

targeted process. It involves the activation of T and B cells, leading to the production

of antibodies and memory cells. This response is crucial for eliminating bacteria and

providing long-term immunity against future infections.

Both multidrug-resistant bacteria and regular bacteria trigger similar immune

responses in the human body after infection. However, one of the reasons multidrug-

resistant bacteria are difficult for the immune system to eliminate is their strong ability

to escape immune surveillance. While regular bacteria also have immune evasion

mechanisms, they are much less potent compared to multidrug-resistant bacteria.

[35]

Figure 2.7 Adaptive response of bacterial response

The immune evasion mechanisms of multidrug-resistant bacteria include, but are

not limited to, the following three aspects:

(1) Virulence factors: Multidrug-resistant bacteria usually produce some virulence

factors, such as exotoxin, endotoxin and protease, which can destroy host cells, interfere

with immune response and inhibit phagocytosis. They can inhibit the inflammatory

response of the host immune system and reduce the ability to recognize and destroy

bacteria.

13

(2) Antigenic variation: Multidrug-resistant bacteria may escape the recognition of host

immune system by changing their surface antigen structure or expressing different

antigens. This antigen variation makes it difficult for the host immune system to

recognize and produce specific immune response, thus making it more difficult for

bacteria to be eliminated.

(3) Biofilm formation: Multidrug-resistant bacteria sometimes form biofilm, which is a

membrane structure composed of multiple layers of polymers, which can protect

bacteria from the attack of host immune system. Biofilm provides a physical barrier to

prevent contact and phagocytosis of immune cells, thus increasing the survival and

replication of bacteria.

The following section will provide a detailed explanation of the immune evasion

mechanisms of multidrug-resistant bacteria.

14

3. Strategies for immune escape of multi-drug

resistant bacteria

3

.1 Multidrug-resistant bacteria can evade immune system

attacks through many strategies

Immune escape refers to a pathogen's ability to successfully elude the host immune

system through a variety of intricate biological pathways, allowing the infection to stay

[36]

within the host organism . The pathogen and the immune system interact dynamically

during this complex process. At first, viruses may alter the way their antigens are

expressed in an effort to avoid being recognized by the immune system. This can be

achieved by controlling or introducing changes in surface antigens, which will make it

difficult for the immune system to identify and launch a suitable defense. Moreover,

pathogens have the potential to deliberately inhibit immune cell function, reducing the

immune cells' capacity to efficiently eradicate infections. Certain diseases secrete

inhibitory chemicals that cause apoptosis in cells or interfere with immune cell

signaling, weakening the immune response. Furthermore, some infections can mimic

the molecular structure of host cell surfaces, making it difficult for the immune system

to distinguish them from the host's own cells. Immune cells are effectively rendered

incapable of precisely identifying and attacking the infections due to this mimicry.

The human immune system has developed to a high degree to combat infections

and neutralize their risks. Multidrug-resistant bacteria have, however, developed a

strategy that successfully evades the host immune system, allowing them to spread

[37]

across several channels and improving their chances of surviving inside host cells

.

Because of this, multidrug-resistant bacteria have developed several tactics to deal with

the host immune system's difficulties and facilitate the easy application of their

pathogenesis, hence increasing the possibility of persistence in the host. These tactics

create a complicated network of numerous approaches and include, among other things,

preventing antigen presentation, imitating molecular structure, isolating antigens,

suppressing the immune system, avoiding autophagy and apoptosis, and adjusting

[38]

costimulatory signals

.

When dealing with germs that are resistant to several medications, the pathogen

has to fight against both the immune system and many drugs at the same time.

Prolonged and incorrect antibiotic use is one of the main factors contributing to the

establishment of multidrug-resistant bacteria. This leads to the slow development of

drug resistance in pathogens. Multidrug-resistant bacteria frequently use a variety of

immunological escape strategies to strengthen their resistance to drugs as well as the

host immune system. Therefore, in addition to treating drug-resistant bacteria, treating

multidrug-resistant bacteria also requires applying targeted therapies to block their

15

immune escape mechanisms. This underscores the intricate and pressing nature of

infectious disease treatment, emphasizing the imperative for global collaboration in

[39]

developing more effective strategies for prevention, control, and treatment

.

3

.2 Suppression of Phagolysosome Maturation

This can be accomplished by inhibiting phagosome acidification, impeding

phagolysosome formation, or suppressing interferon production. It's noteworthy that,

while interferon is not typically considered for direct use against bacteria, as it is

commonly believed to be effective against viral replication and transmission, interferon

can indirectly eliminate multidrug-resistant bacteria that invade the host by stimulating

the activity of immune cells such as macrophages and natural killer cells. Inhibiting

interferon synthesis is a natural strategy for multidrug-resistant bacteria attempting to

[40]

evade immune clearance

.

A common strategy employed by multidrug-resistant bacteria is inhibiting the

formation of the phagolysosome to avoid exposure. Acidification is crucial for the

survival of bacteria inside the phagosome. The V-ATPase pumps protons into the

phagosome to increase its acidity, marking phagosome maturation. However, certain

bacteria release a protein known as PtpA (protein tyrosine phosphatase), which binds

to the H subunit of V-ATPase, inhibiting ATP activity and ultimately hindering the

acidification process (figure 1).

Figure 3.1 PtpA hinders the acidification process

The second approach involves inhibiting the fusion of the phagosome and

lysosome. Phagosomes, marked by Rab5 proteins, exchange Rab5 for Rab7 to fuse

with lysosomes. Some bacteria recruit Rab22a proteins, preventing this exchange and

resulting in the inhibition of phagolysosome formation. Another protein, TACO, present

16

on the phagosome, detaches to deliver the phagosome towards the lysosome. However,

in the case of certain bacteria, the TACO protein is retained on the phagosome, delaying

[41]

the fusion of the phagolysosome

.

The third method is the inhibition of interferon production. Within macrophages,

CGAS molecules detect foreign DNA, activating the STING pathway, and ultimately

ensuring interferon production. However, without degradation, no foreign dsDNA

from bacteria is exposed to CGAS, leading to no detection and consequently no

[42]

interferon production

.

3

.3 Escape from Oxidative Killing by Neutrophils

One of the two ways that multidrug-resistant bacteria evade immune system attack

is that they can easily evade the oxidative killing of neutrophils, causing neutrophils to

die. Mycobacterium tuberculosis, renowned for its capacity to exhibit multidrug

resistance, presents a formidable challenge in infection scenarios. Neutrophils,

operating in an oxidative fashion at the infection site, play a pivotal role in eliminating

pathogenic bacteria. However, the survival of multidrug-resistant (MDR) bacteria

coincides with the necrotic cell death of infected neutrophils, a process intricately

linked to the production of radical oxygen species (ROS).

Figure 3.2 RD1 causes free radical products to be released into the cytoplasm

Region of Difference 1 (RD1) emerges as a pivotal factor in the pathogenicity and

immune escape capabilities of Mycobacterium tuberculosis. Within the RD1 region,

several genes, notably EsxA and EsxB, stand out as key players involved in both cell

lysis and immune escape. The proteins encoded by EsxA and EsxB are secreted,

forming a complex known as the "ESX-1 secretion system," emphasizing their integral

role in processes such as cell lysis (figure 2). In the context of neutrophil lysis,

17

Mycobacterium tuberculosis strategically releases EsxA and EsxB proteins through the

ESX-1 system, inducing the formation of pores that rupture the membrane structure.

The presence of RD1 disrupts the membrane, leading to the release of radical products

(ROS, NADPH Oxidase, MPO) into the cytosol, ultimately resulting in necrosis. This

interconnected series of events underscores the bacterium's sophisticated survival

mechanisms within the host and highlights the challenges inherent in combating its

[43]

multidrug resistance and immune evasion tactics

.

3

.4 Neutralization of antimicrobial peptides (AMPs) in

multidrug-resistant bacteria

Some multidrug-resistant bacteria possess the ability to suppress the innate

immune response by inhibiting the functionality of antimicrobial peptides (AMPs).

AMPs, a class of small proteins naturally occurring in living organisms, exhibit a broad

spectrum of capabilities in combating various microbial infections within the immune

system. These peptides operate through diverse mechanisms. Firstly, they exert a direct

bactericidal effect, disrupting pathogen membranes and interacting with nucleic acids

and proteins, ultimately leading to microbial death. Secondly, AMPs play a role in

immune regulation, influencing immune system activity. They activate and enhance the

function of immune cells, such as macrophages and neutrophils, improving the host's

[44]

immune response against pathogens and balancing the inflammatory response

.

Figure

3

.3 Antimicrobial peptides (AMPs)

AMPs serve as effector molecules of innate immunity, constituting the body's

initial defense against invading microorganisms. Characterized by their short cationic

nature and amphiphilic properties, AMPs can interact with both hydrophobic and

[45]

hydrophilic molecules . The initial electrostatic interaction between AMPs and target

organisms is facilitated by the positive charge of AMPs and the anionic nature of

bacterial surfaces, allowing them to attract and combine easily. In some multidrug-

resistant bacteria, increased lysX expression is associated with alterations in the cell

18

surface's negative charge. The lysX gene catalyzes lysine transfer to

[46]

phosphatidylglycerol, resulting in an overall positive charge on the cell membrane

.

Consequently, antimicrobial peptides fail to interact with invading microbes as

effectively, leading to a diminished innate immune response and enabling multidrug-

resistant bacteria to successfully evade immune surveillance (figure 3).

3

.5 Manipulating Host Cell Responses for Immune Evasion

Multi-drug resistant strains of bacteria employ a strategy to evade immune cell

attacks by modulating host cell apoptosis and autophagy, influencing the overall

immune response. Taking Mycobacterium tuberculosis (Mtb) as an example of a multi-

drug resistant pathogen, its virulence is intricately linked to the regulation of host cell

apoptosis. Highly virulent strains can suppress both cellular autophagy and apoptosis,

leading to latent infection and immune escape. Various components, including miR-

3

0A, play roles in Mtb virulence while simultaneously affecting the apoptotic response

of macrophages. The overexpression of miR-30 inhibits autophagy, contributing to Mtb

persistence. Mtb invasion and induction of host cell apoptosis involve numerous

cellular factors and signaling proteins, impacting pathways such as TNF-α. NKT cells

contribute to inhibiting Mtb growth in macrophages through IFN-γ production.

Macrophages and dendritic cells are crucial components of the initial defense against

Mtb, relying on cellular immunity. T cell activation is a fundamental aspect of the

immune response to Mtb, with Th1 cells playing a protective role through the secretion

of IL-2 and IFN-γ. The comparable proportion of T cells expressing γ/δ T cell receptors

in tuberculosis patients and controls indicates their involvement in the early immune

response. Mtb-infected macrophages exhibit outcomes of necrosis, apoptosis, or

survival. Apoptosis serves as a primary defense strategy against Mtb, presenting

antigens to dendritic cells and controlling bacterial reproduction. Autophagy, associated

with specific genes, contributes to homeostasis and immune responses. TNF-induced

macrophage apoptosis is like Mtb-induced apoptosis, distinct from non-apoptotic

complement-promoted cell death, which has no effect on bacterial activity. In summary,

the inhibition of autophagy and apoptosis by highly virulent Mtb can lead to latent

infection and immune evasion^[⁴⁷^]

.

3

.6 Interplay with Antigen Presentation Dynamics and the

Role of Antigen Export

Antigen presentation is a key process in the immune system, where specialized

antigen-presenting cells break down phagocytic antigens internally into small

fragments, then bind these fragments to major histocompatibility complex (MHC)

molecules, and finally display them on the cell surface. MHC-I mainly presents

antigens produced by infected cells, while MHC-II mainly presents exogenous antigens,

[48]

such as bacteria and viruses . T lymphocytes recognize these MHC-antigen

19

complexes through T cell receptors and activate corresponding immune responses,

including cytotoxic immunity and humoral immunity, thereby protecting the body from

infection and disease(figure 4). Mycobacterium tuberculosis (Mtb) uses many ways to

evade the host immune system and survive in the host environment. After entering

dendritic cells (DCs), Mtb coordinates its movement to nearby lymph nodes, where a

significant reduction in CD4 T cell activation reveals the bacteria's superb control over

antigen presentation dynamics. This situation is further complicated by the fact that Mtb

antigens tend to move extracellularly from infected DCs to uninfected counterparts,

evading the infected cells' antigen presentation inhibition. Extensive research suggests

that Mtb deliberately strays bacterial pathogens from the antigen presentation route in

[49]

the early phases of antigen export and transfer

.

Figure 3.4 Antigen presentation

The activation of antigen-specific CD4 T cells by infected cells is enhanced by the

depletion of kinesin-2, which grants a complex level of control over intracellular

infection. Through a convincing nexus, Mtb-infected DCs and macrophages redirect

bacterial antigens from the MHC II antigen-presenting pathway to the extracellular

milieu, further undermining host immune responses. This strategic relocation

diminishes CD4 T cell activation, fostering an environment conducive to heightened

intracellular bacterial survival. This intricate dance between Mtb and the host immune

system highlights the adaptability and resilience of the bacterium in circumventing

[50]

immune surveillance . Of particular significance is the emergence of antigen export

as a paramount strategy for immune evasion, whereby Mtb ensures its sustained

[51]

survival notwithstanding the development of an antigen-specific T-cell response

.

This nuanced exploration illuminates the multifaceted strategies employed by Mtb to

subvert the host immune machinery, offering insights into potential avenues for

therapeutic intervention.

20

3

.7 Escape from the phagosome before the fusion begins

Certain multidrug-resistant bacteria display a shrewd evasion strategy, skillfully

evading the phagosome before fusion takes place. This intricate maneuver, notably

observed in pathogens like Shigella(figure 5), bestows upon them a strategic advantage

[52]

in infiltrating host cells . Following their successful escape into the host cell's

cytoplasm, these bacteria capitalize on this tactical evasion to initiate a cascade of

unbridled proliferation. Remarkably, the capacity of these bacteria to form actin tails

emerges as a multifaceted tool, not only facilitating seamless intracellular movement

but also empowering them to traverse adjacent cells. This dynamic capability

perpetuates the infectious cycle with remarkable efficiency, allowing the bacteria to

propagate through host tissues. Moreover, this sophisticated evasion mechanism

ensures that these microbes continue their propagation without ever exposing

[53]

themselves to the extracellular milieu . By strategically avoiding contact with the

external environment, where they would be susceptible to attacks by the immune

system, these bacteria maintain a covert and resilient presence within the host. This

astute adaptation underscores the remarkable resilience and versatility of MDR bacteria,

highlighting their ability to exploit sophisticated evasion strategies for sustained

survival and propagation within the host environment.

Figure 3.5 Shigella and actin tails

21

4. Approaches for the treatment of infections due

to multidrug-resistant bacterial pathogens

This chapter will focus on introducing approaches related to host immunity and

briefly introduce other methods.

The current pace of antibacterial drug development is insufficient to tackle

AMR.Out of 68 clinical development projects enlisted in the WHO antibacterial

[54]

pipeline , a majority (41 projects) are based on traditional approaches involving small

molecules (antibiotics and different combinations of antibiotics), among which only 18

(

44%) molecules fulfill at least one of innovation criteria for antimicrobial drugs (new

[55]

target, absence of known cross-resistance, unique mode of action or new class) . The

rest of all ongoing clinical development projects and almost all newly approved

antibiotic molecules are based on the same scaffold discovered four decades ago.

[56]

Figure 4.1 Clinical Antibacterial Pipeline

Finding newer antibacterial molecules with novel scaffolds using traditional

approaches seems nearly impossible as all the low-hanging fruits have already been

picked. Thus, the need of the hour is to look toward some unconventional approaches.

22

Figure 4.2 Approaches for the treatment of infections due to

multidrug-resistant bacterial pathogens

4

.1 Antimicrobial peptides-based therapies

The first novel approach is Antimicrobial peptides-based therapies.Host defence

peptides (HDPs) are an evolutionarily ancient component of the innate immune system

of mostmulticellular organisms.^[They exhibit a wide range of biological activities

from direct killing of invading pathogens to modulation of immunity and other

biological responses of the host. Two major families of naturally occurring HDPs have

been distinguished: defensins and cathelicidins.

57]

The evolutionarily widespread distribution and the extreme diversity of HDPs

highlight their prominent role in immune defences. Historically, this has been attributed

to their antimicrobial activity but in recent years potent immuno-modulatory properties

of HDPs have been characterised and suggested to be an important part of their

[58-59]

biological function.

23

Table 4.1 The diversity of mammalian host defence peptides

A wide range of HDPs from different species have been shown to act as

chemoattractants for cells of innate and adaptive immunity. As an example, human

cathelicidin LL-37 attracts neutrophils, monocytes, T cells, and mast cells,using formyl

[60-61]

peptide receptor-like 1 (FPRL1), and a distinct Gi-coupled receptor.

Figure 4.3 Immunomodulatory activity of host defence peptides:human cathelicidin LL-37.

During recent decades, several hundred publications have documented the direct

antibiotic effects of the AMPs; therefore, it is not surprising that the physiological role

of the peptides has been considered to be primarily antimicrobial.However, it has

become increasingly clear that many of these peptides, in addition to their direct

24

antimicrobial activity, also have a wide range of immunomodulatory properties.

Figure 4.4 Besides having direct antimicrobial effects, defensins posses a variety of additional

functions related to host defense.

Table 4.2 Sequence and localization of the human defensins

Host defense peptides induce or inhibit the release of an array of cytokines and

chemokines from a variety of different cell types.Members of the prostaglandin and

leukotriene family are agents that participate in the rapid induction of an inflammatory

25

response by increasing vascular permeability and thus help to establish the

[62]

inflammatory infiltrate

.In addition to direct activation of innate host defense

mechanisms, some PRRs are coupled to the induction of adaptive immune

responses.An ever-expanding palette of both immune and nonimmune properties has

been ascribed to the defensins. Defensins also play a role in wound healing,

[63]

proliferation of epithelial and fibroblast cells, angiogenesis and vasculogenesis.

4

.2 Phage-based therapies

Bacteriophages are anobligate parasites that naturally infects bacteria and destroy

their bacterial host upon infecting them. Phages are very specific to their host and have

recently emerged as a novel therapeutic choice as it can successfully kill several

[63-66]

pathogens either alone or in combination with antibiotic therapy

. Owing to the

limitations posed by phage biology, recent advances in phage therapy include using

bioengineering methods to develop potential phage therapeutics and their purified lytic

[66]

proteins . Phage therapy has shown great potential

in the treatment of GNB biofilm-forming bacteria both under in vivo and in vitro

conditions.

One of the major applications of bacteriophages has been their utilization for the

treatment of MDR and XDR Tuberculosis (MDR-TB and XDR-TB) for which there are

extremely limited therapeutic options available. From this perspective, several research

[65]

groups have investigated bacteriophages as an alternative to antibiotics

.

4

.3 Immunomodulatory therapy

Another viable alternative is the use of immunomodulatory therapies against

infections caused due to drug-resistant pathogens. One of the foremost examples is

adjuvant immunotherapy against drug-resistant TB. Ad-juvant immunotherapy

includes host-directed immunomodulation with immuno-adjuvants, use of recombinant

cytokines, corticosteroids and vaccination as promising antimicrobial therapies.

As we can see in the figure 4.5, overt immune responses characterise the

pathological outcome in tuberculosis (TB). Neutralisation of pro-inflammatory

cytokines such as IL-6, TNF-α, VEGF and IFN-αβ, as well as anti-inflammatory IL-4

during severe pulmonary disease may help reduce ongoing parenchymal damage in the

lung. Alternatively, suboptimal activation of anti-TB immune responses due to

regulatory T cell activity can be reversed by the use of the anti-cancer drug

cyclophosphamide. Drugs with anti-TB potential, such as metformin, imatinib,

ibuprofen, zileuton, valproic acid, and vorinostat as well as nutraceuticals such as

vitamin D3 not only abate bacterial burden via host-dependent mechanisms, but may

also fine-tune the immune response to Mycobacterium tuberculosis (M. tb). These

26

drugs increase phagocytosis of extracellular bacteria, improved emergency myeloid

response and increased autophagic and apoptotic killing of bacteria, subsequently

editing the T cell response in favour of the host. Immune checkpoint inhibition with

blockade of the PD-1/PD-L1,CTLA-4, LAG3 and TIM3 pathways may improve the

[67]

quality of the cellular immune response to M. tb epitopes, as seen in cancer.

Figure 4.5 Host-directed therapies aimed at modulating immune responses

in the tuberculous lung.

4

.4 Antibody-mediated therapy

In combating AMR, pathogen-specific monoclonal antibodies (mAbs) can act as a

promising alternative therapy and it has potential to reduce antibiotic utilization

worldwide. Owing to the recent advancements in molecular biology and antibody

therapy, it is possible to generate systematic, homogenous, fully human, and/or

humanized mAbs by using single antigen-specificity to target the desired pathogen.

This process requires only a pathogen antigen (immunogen) and an immunized

individual for the successful development of humanized antibody.

Antibody-mediated therapies have targeted various exotoxins, bacterial epitopes

27

and virulence factors. In the past, monoclonal antibodies against several pathogens likes

Streptococcus pyogenes, Clostridium and Escherichia coli have been tested at different

[68]

clinical stages.

4

.5 Macrophage cell therapy

Hematopoietic cells are endowed with a variety of mechanismsto control

microbial invasion. Macrophages in particular have long been appreciated as potent

antimicrobial immune cells equipped with several receptors that allow for rapid

recognition,phagocytosis, and killing of pathogenic microbes, coupled to secretion of

immunostimulatory cytokines to further orchestrate a robust multifaceted antibacterial

immune response.

Deborah G. demonstrated that ICONIMAC^T^Mcan be generated through a simple

and good manufacturing practice (GMP)compatible process, in which cells can be

cryopreserved and delivered “off the shelf.” Both allogeneic and xenobiotic

TM

ICONIMAC macrophages rescued immune-competent mice from infection-induced

lethality, without inducing an excessive inflammatory response or graft versus host

disease (GVHD).Data from this study support the continued development of

macrophage-based cell therapy strategies to treat antibioticresistant bacterial

[69]

infections.

4

.6 Targeting unconventional targets

SrtA are cysteine trans-peptidases that help in building the cell wall architecture

and attachment of cell wall proteins.These are found ubiquitously in GPB, while

genomic data analysis has revealed the presence of sortase superfamily genes in GNB

and archaeal species.In GPB, the role of sortase is well known, but in GNB and archaeal

species, it is not that clear.

In GPB, cell wall anchored surface protein contains a highly conserved C-terminal

LPXTG motif, these cell surface proteins have shown a role in host-pathogen

interactions and can act as a virulence factor, thus can be a potentially good target for

future drug discovery research.

4

.7 Allosteric target

Most of the antibiotics target the active site of the enzymes called orthosteric

inhibitors. However, the biological activities of enzymes can also be regulated by

allosteric modulators. Allosteric modulators are the compounds that bind other than at

the active site of biocatalyst.

28

In 2021, Mayland Chang et al. studied a novel allosteric inhibitor ceftaroline that

[70]

has shown anti-MRSA activity in screening as well as docking analysis. Some other

example of novel allosteric modulators includes quinazolinones and PBP2a in case of

S.aureus and aminoglycoside-modifying enzymes (AMEs) including acetyltransferase,

[71]

phosphotransferase and nucleotidyl transferase. To date, PBP2a (penicillin-binding

protein 2a) is a promising bacterial protein target molecule for the novel quinazolinone

[72]

class as well as oxadiazoles class by allosteric inhibition.

4

.8 Conclusion

The current drug development strategies toward drug resistance pathogens need to

[73]

evolve faster to reduce the global burden of AMR . The problem of drug resistance

is getting worse day by day; thus, there is ample scope to utilize multiple avenues of

therapies to combat the unrelenting tide of AMR.

AMR is one of the major concerns to human health worldwide, and these drug

resistant pathogens need our urgent attention. Whereas traditional approaches, i.e.,

developing newer antimicrobial drugs, appear insufficient to battle these superbugs. We

need to explore novel and alternative strategies along with conventional approaches to

fight these hard-to-deal with pathogens. AMR cannot be eradicated by only developing

new drugs; alternative approaches to combat drug-resistant pathogens are equally

important.

29

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