Carbapenem-Resistant Klebsiella pneumoniae (CRKP)

Table of Contents

• What is Klebsiella pneumoniae?
• Morphology of Klebsiella pneumoniae
• Biochemical Characteristics of Klebsiella pneumoniae
• Cultural Characteristics of Klebsiella pneumoniae
• Virulence Factors of Klebsiella pneumoniae
• Clinical Manifestation of Klebsiella pneumoniae
• What are Carbapenems?
• What are Carbapenem-Resistant Organisms (CROs)?
• What is Carbapenem-resistant Klebsiella pneumoniae (CRKP)?
• Risk Factors for Carbapenem-resistant Klebsiella pneumoniae Infections
• Mechanisms of Resistance Against Carbapenems by CRKP
• a. Enzymatic Modification of Antibiotics
• b. Modification of Porins
• c. Over Expression of Efflux Pumps
• d. Capsule
• e. Biofilm Formation
• Epidemiology of Carbapenem-resistant Klebsiella pneumoniae
• Identification of Carbapenem-resistant Klebsiella pneumoniae
• Biochemical Algorithm For Identification of Klebsiella pneumoniae
• Confirmation of Carbapenem Resistance in Klebsiella pneumoniae (CRKP)
• Molecular Confirmation of CRAB
• Prospective Treatment Options for CRKP Infections
• Future/Alternative Treatment Options Under Study
• Prevention and Control Measures from CRKP Infections

What is Klebsiella pneumoniae?

In the family Enterobacteriaceae of the phylum Pseudomonadota, Klebsiella pneumoniae is a Gram-negative, facultatively anaerobic, rod-shaped, non-motile, lactose-fermenting, encapsulated Gammaproteobacteria. It belongs to the genus Klebsiella and is a pathogenic species. It is also known as "Friedlander's Bacillus" since German microbiologist and pathologist Carl Friedlander initially identified it in 1882.

A genus of rod-shaped, encapsulated, Gram-negative, oxidase-negative bacteria called Klebsiella belongs to the family Enterobacterales. The genus Klebsiella now contains seven well characterised species, some of which are further divided into subspecies. All of species are common and are mostly found as commensals in the intestines of animals. They can also be found in sewage, rotting organic waste, decaying plants, polluted soil, contaminated water, etc. Four species, nevertheless, are usually cited as being harmful to humans. K. rhinoscleromatis, K. oxytoca, K. aerogenes, and K. pneumoniae. 

K. pneumoniae is the most significant human pathogen of them. Although it is categorised as an opportunistic pathogen, it is the pathogen of this genus that has been recorded the most frequently, notably infecting people who have been admitted to hospitals and having a high rate of morbidity and death. It is mostly to blame for respiratory tract infections that patients get when hospitalised. While still prevalent, community-acquired RTIs caused by K. pneumoniae are less common than nosocomial RTIs. Immune-compromised individuals and those who contract antibiotic-resistant strains may have death rates of up to 50%, while cases with alcoholism and bacteremia may see rates as high as 100%.   

Additionally, K. pneumoniae has a very high rate of fast development of antibiotic resistance against current choices. Several bacteria are currently resistant to carbapenems, the last-resort antibiotic. Additionally, because of its ability to create a capsule and a biofilm, it may live and colonise on dry surfaces like inanimate objects and medical equipment, which makes it possible for it to infect individuals who are immunosuppressed and in hospitals. And this is causing K. pneumoniae infection case severity and fatality rates to rise.     

K. pneumoniae plays a crucial function in agricultural productivity in addition to having a detrimental impact on human health. Numerous studies have demonstrated their ability to fix nitrogen in soil that is anaerobic. 

Morphology of Klebsiella pneumoniae

• Rod-shaped Gram-negative
• generally arranged individually, but sometimes occasionally found in pairs, short chains, and clusters
• 0.5 to 0.8 by 1.0 to 2.0 microns 
• Non-motile
• Almost all nonvirulent strains are highly capsulated;
• Non-sporing
• Non-flagellated

Biochemical Characteristics of Klebsiella pneumoniae

• Anaerobic and Facultative anaerobe 
• Lactose fermentative
• Oxidase negative
• Catalase positive
• IMViC test = -ve, -ve, +ve, +ve  (- – + +)
• Urease positive
• Nitrate positive
• TSI test = Alkaline/Alkaline (Red/Red), gas production, no H2S

Cultural Characteristics of Klebsiella pneumoniae

• Optimal temperature for anaerobic or facultative incubation is 35 (2)°C, with a temperature range of 15–40°C. 
• Although it can survive in a slightly alkaline pH range and can't grow at acidic pH, the optimal pH need is 7.0.
• Without a strict dietary regimen
• In laboratories, nutrient agar and MacConkey agar are frequently employed.

Nutrient Agar = small 2 – 3 mm, dome-shaped, circular, Smooth (in fresh culture), Mucoid, greyish white, Opaque – Translucent colonies 

MacConkey Agar = small 2 – 3 mm, convex, lactose fermentative, circular, Smooth, Mucoid, Opaque, Red or Pink colonies

Blood Agar = small 2 – 3 mm, non-hemolytic, greyish white, Smooth, Mucoid, Translucent – Opaque colonies

Eosin Methylene Blue Agar = small 2 – 3 mm,  convex, smooth, mucoid, Pink or purplish, Opaque – Translucent colonies

Virulence Factors of Klebsiella pneumoniae

Numerous extracellular and cell-associated virulence factors are present in K. pneumoniae, which contribute to its pathogenicity. The following table provides a summary of the key K. pneumoniae virulence factors and their suggested functions.

Virulence Factors of Klebsiella pneumoniae and Roles

Capsule

Outside of the bacterial cell wall, it is the outermost polysaccharide layer enveloping the bacterial cell. 

• Helps in protecting bacterial cells by preventing phagocytosis.
• Promotes the adhesion and development of biofilms
• Shields the invading bacteria from the host's complement system's deadly effects
• Provide immunity against a number of antibodies and antimicrobial peptides
• Create an antibiotic resistance.
• Rarely target and inhibit immune cells
• Functions as a physical barrier to chemical and physical stressors

Fimbriae

They are tiny projections that promote bacterial colonisation and adherence. 'Type 1' and 'Type 3' fimbriae are significant virulence factors.

• Type 1 fimbriae are in charge of cell attachment and the development of bacterial communities.
• Type 3 fimbriae are in charge of biofilm development and aid in bacterial adhesion to host cells and medical equipment.

Porins

They are protein channels in the outer membrane of bacteria that control their permeability. The most significant porins produced by K. pneumoniae are OmpK 35, OmpK 36, and OmpK 37. 

• OmpK 36 is discovered to interfere with complement system activation, allowing bacteria to evade the host's immunological response.
• Promotes biofilm development and attachment
• provide antibiotic resistance

Lipopolysaccharides (LPS) 

LPS are responsible for:

• Adherence
• Resistance against phagocytes
• Activation of the complement factors and protect the bacteria against the host’s complement killing
• Conferring serum resistance to the bacteria
• Helps in colonization and dissemination inside the host’s internal organs
• Helps in the formation of biofilm
• Confer antibiotic resistance 

Outer Membrane Proteins (OMPs)

They are transport-related outer membrane protein vesicles that the bacterium's outer membrane secretes. 

OMPs are responsible for:

• Preventing activation of epithelial cells in the host’s respiratory tract and protecting from the inflammatory responses
• Protects against neutrophils
• Acquisition of antibiotic resistance

Siderophore

The bacterial cell has a mechanism in place to satisfy its need for iron.

• Promotes bacterial iron acquisition and survival inside the host’s body

Type 6 Secretory System

• Helps in injecting effector proteins inside the host’s cells to destroy the host cells and limit their actions against the invading bacterial cells

Clinical Manifestation of Klebsiella pneumoniae

Normal flora of the mouth, digestive system, and occasionally skin includes K. pneumoniae. It causes both community- and hospital-acquired infections, although the rate of hospital-acquired infections is significantly greater. Along with pneumoniae, K. pneumoniae is described as a common pathogen that may infect the blood, meninges, liver, wounds, and urinary tract. It is a well-known pathogen that causes more than 10% of HAIs.   

The development of biofilms and resistance to the majority of the current antibiotics have raised the morbidity and death rates associated with K. pneumoniae infections. 

Frequently connected clinical symptoms with K. pneumoniae include:

1. Respiratory Tract Infections (Pneumonia)

After Streptococci, K. pneumoniae is the most prevalent bacterial pathogen causing pneumonia. About 12% of hospital-acquired pneumonia is attributed to it. It causes pneumonia in patients in critical care units (ICUs) as well as pneumonia associated with ventilators (VAP). Additionally, it is mostly to blame for community-acquired pneumonia, especially in alcoholics. The distinctive mass of sputum known as "currant jelly" is produced in K. pneumoniae pneumonia, which causes necrosis of the lung tissue in addition to producing mucus, cell debris, and blood in sputum.  

2. Urinary Tract Infections

Another typical illness brought on by K. pneumoniae is UTI, which is more frequent in hospitalised patients with indwelling urinary catheters. Patients with renal impairment and catheterized patients are particularly vulnerable to K. pneumoniae-caused UTIs. UTIs obtained in the community are also often reported. About 6–17% of all UTI cases are related to K. pneumonia. 

3. Soft Tissue and Skin Infection

Surgical wounds and wounded skin are known to be infected with K. pneumoniae. Myositis, necrotizing fasciitis, and cellulitis are the outcomes. Additionally, they are said to damage the lining of the oesophagus, stomach, and intestines.  

4. Blood Stream Infection (BIs) and Septicemia

4–15% of instances of septicemia and 3–20% of newborn septicemia are caused by K. pneumoniae. Studies have revealed that secondary bacteremia and septicemia are more frequent than initial ones in Klebsiella pneumoniae infections because the bacteria is mostly transmitted from the lung to the circulation. When individuals are infected with resistant strains or have additional underlying medical problems, the fatality rate may exceed 70%.    

5. Central Nervous System Infections (Meningitis)

Patients who are hospitalised and have underlying medical problems including diabetes and liver cirrhosis frequently develop Klebsiella pneumoniae-associated meningitis.

6. Pyogenic Liver Abscess

People with diabetes, alcoholism, and those who have received extended antibiotic medication are more likely to get it.  Along with edoema and inflammation in the surroundings, pus forms inside the liver. It even has the potential to harm the liver if untreated. 

7. Endophthalmitis 

Endophthalmitis can potentially result in irreversible blindness in patients with K. pneumoniae BIs.

What are Carbapenems?

• Broad-spectrum β-Lactam antibiotics are used against serious infections, usually against those caused by multidrug-resistant bacterial pathogens. 
• Can resist the effects of β-Lactamase enzymes
• 'Carbapenemases' susceptible 
• Beta-lactam antibiotics feature a double bond between the C-2 and C-3 of the beta-lactam ring, with the side chains organised in the trans position, and sulphur at the C-1 position.
• Binding to penicillin-binding proteins, which prevents bacteria from forming cell walls, is the mode of action.
• IMIPENEM, MEROPENEM, ERTAPENEM, and DORIPENEM are the four commonly used carbapenems.
• referred to as "the antibiotics of last resort"

What are Carbapenem-Resistant Organisms (CROs)?

• Bacteria that have evolved one or more defences against carbapenems are known as carbapenem-resistant organisms. 
• They have the designations of "priority one pathogen" by the World Health Organisation (WHO) and "urgent threat organisms requiring urgent countermeasures" by the US Centres for Disease Control and Prevention (CDC). 

Among the most dangerous bacteria resistant to carbapenem are:

1. Carbapenem-resistant Enterobacteriaceae (CRE); the most significant of them is carbapenem-resistant Klebsiella pneumoniae.
2. Carbapenem resistant Acinetobacter baumannii (CRAB)
3. Carbapenem resistant Pseudomonas aeruginosa (CRPA)

What is Carbapenem-resistant Klebsiella pneumoniae (CRKP)?

The Klebsiella pneumoniae bacteria known as carbapenem-resistant Klebsiella pneumoniae (CRKP) have evolved a number of defence mechanisms to fend off the inhibitory or bactericidal effects of carbapenems.

• The CRE group's member who expresses the most concern is CRKP. CRE is classified by the US CDC as "an urgent threat" for 2019 and by the WHO as "a priority 1 - critical pathogen" since 2017. CRKP is the most significant pathogen in the group with a significant hazard to the general public. It belongs to the ESKAPE group, which also includes MDR pathogenic bacteria linked to the majority of HAIs. 
• Compared to infections brought on by K. pneumoniae sensitive strains, CRKP infections had greater case severity and fatality rates. A mortality rate of 23% to 75% is possible. 

Risk Factors for Carbapenem-resistant Klebsiella pneumoniae Infections

• Patients in hospitals and those with weaker immune systems
• Patients having urinary catheters in place who are catheterized 
• patients requiring ventilation and intubation
• Patients who use steroids, anti-pseudomonal penicillins, have a cardiac ailment, diabetes mellitus, chronic renal disease, urinary blockage, or are receiving long-term antimicrobial medication
• Transplant recipient
• recent surgical patients and open wound patients
• Patients with Chronic Obstructive Pulmonary Disease (COPD)
• Acute and chronically sick patients

Mechanisms of Resistance Against Carbapenems by CRKP

K. pneumoniae has created a number of defences against carbapenems. Among the extensively researched resistance mechanisms are:

a. Enzymatic Modification of Antibiotics

The carbapenemase enzymes that CRKP can manufacture can hydrolyze the carbapenems' β-lactam ring, giving them tolerance to the harmful effects of carbapenems. 

Below is a list of the carbapenemases that K. pneumoniae has been identified to generate. 

• Only CRKP reports the "K. pneumoniae carbapenemase (KPC) β- lactamases". The primary method used by CRKP to circumvent the effects of carbapenems is the development of KPC-lactamases. now North Carolina, USA, initially detected K. pneumoniae in the late 1990s, it has now been reported from all over the world. The most frequently seen β-lactamases in K. pneumoniae are of the KPC-2 and KPC-3 types. 
• The “Metallo –β – Lactamases (MBLs)” are another important carbapenemases produced by many of the clinical isolates of K. pneumoniae. New Delhi Metallo – β– Lactamase – 1 (NDM – 1) , Imipenem-resistant Pseudomonas (IMP) Metallo-β-lactamase group and Verona integron-related Metallo-β-lactamase (VIM-1 and VIM-2) groups are the most frequently reported MBLs types in CRKP.
• K. pneumoniae also produces class-D β-lactamases such "Oxacillinase (OXA-β-lactamase)," mostly of the OXA-48 type and its variants OXA-184, OXA-204, and OXA-232.

b. Modification of Porins

The outer membrane porins OmpK35, OmpK36, and OmpK37 have undergone modification, according to CRKP. In CRKP strains, OmpK35 is severely downregulated and either scarcely present or totally missing. 

c. Over Expression of Efflux Pumps

Another method K. pneumoniae developed to remove carbapenems from their cytoplasm and impart resistance to them is over-expression of the efflux pumps. The most significant efflux pump family that contributes to resistance to carbapenems is the RND type efflux pump family. AcrAB and OqxAB are the most effective efflux pumps among the RND kinds. 

d. Capsule

By serving as a physical barrier and dilution agent for the antibiotics, the acidic polysaccharide capsule of K. pneumoniae also helps a bit in imparting resistance against carbapenems. 

e. Biofilm Formation

Another common mechanism that imparts resistance to several antimicrobials, including carbapenems, is biofilm development. It functions as a physical obstacle that keeps carbapenems out of the cell.

Epidemiology of Carbapenem-resistant Klebsiella pneumoniae

Since the early 21st century, when it made its initial report in the Northern USA, CRKP has become a worldwide problem. K. pneumoniae strains resistant to carbapenem have become widespread pathogens causing HAIs over the past ten years. There is an endemic incidence of CRKP in the USA, Latin America, South American nations, European nations, Southeast Asian nations, China, Mediterranean nations, and even African nations. In endemic areas, their incidence in hospital settings ranges from 20 to 40%.

Identification of Carbapenem-resistant Klebsiella pneumoniae

In diagnostic and research facilities, the phenotypic identification technique is frequently employed. Molecular techniques are employed for more accurate identification (studying processes and genetics) and research projects. 

The isolate is first identified as K. pneumoniae in the laboratory using a biochemical testing procedure, and then it is examined for carbapenem resistance.  

Biochemical Algorithm For Identification of Klebsiella pneumoniae

• Gram-negative Rod
• Non-motile
• Capsulated
• Oxidase – ve 
• IMViC = – – + +
• Nitrate +ve 
• Urease reducing (+ ve) 
• TSI = Y/Y (Acidic/Acidic), Gas + ve and H2S –ve
• Growth on KCN +ve 

Confirmation of Carbapenem Resistance in Klebsiella pneumoniae (CRKP)
Phenotypic Confirmation of CRKP by Antimicrobial Sensitivity Testing and Calculation of Zone of Inhibition

By utilising carbapenems for an antimicrobial sensitivity test, it is possible to validate the phenotype of CRKP. We can establish the isolate is CRKP if the zone of inhibition is less expansive than suggested or exhibits higher levels of resistance.Presented here is the zone size interpretation chart for carbapenems against K. pneumoniae in accordance with the CLSI standard.

	Zone Size (in mm) For:	Zone Size (in mm) For:	Zone Size (in mm) For:
Carbapenem Antibiotics	Sensitive	Intermediate	Resistance
Doripenem (DOR 10 mcg)	≥23	20 – 22	≤19
Ertapenem (ETP 10 mcg)	≥23	19 – 21	≤18
Imipenem (IMP 10 mcg)	≥23	20 – 22	≤19
Meropenem (MRP 10 mcg)	≥23	20 – 22	≤19

The chart below shows the zone size interpretation for carbapenems against K. pneumoniae in accordance with the EUCAST standard.

	Zone Size (in mm) For:	Zone Size (in mm) For:	Zone Size (in mm) For:
Carbapenem Antibiotics	Sensitive	Intermediate	Resistance
Doripenem (DOR 10 mcg)	≥22	17-21	≤16
Ertapenem (ETP 10 mcg)	≥25	–	≤25
Imipenem (IMP 10 mcg)	≥22	18-21	≤17
Meropenem (MRP 10 mcg)	≥22	17-21	≤16

The common methods for phenotypic detection of carbapenemase production in CRKP include:

1. Modified Hodge Test
2. Carba NP Test
3. Modified Carbapenem Inactivation Method / Carbapenem Inactivation Method
4. Blue Carba Test
5. EDTA Inhibition Method
6. Boronic Acid Inhibition Test
7. mCIM/eCIM Test
8. Double Disk Synergy Test (using 10 μg 0.5 M EDTA disk and 10 μg imipenem or meropenem disk) and Combined Disk Synergy Test (using one imipenem or meropenem disk with EDTA and another without EDTA) 
9. Chromogenic media: currently two chromogenic agar mediums are used to separate carbapenemase producers from non-producers, they are: CHROMagar-KPC, and Brilliance CRE agar.

Molecular Confirmation of CRAB

1. Polymerase Chain Reaction (PCR) 
2. Xpert Carba-R molecular test 
3. Multiplex PCR 
4. Duplex multiple cross displacement amplification combined with lateral flow biosensor (MCDA-LFB) method 
5. DNA Microarray 
6. Verigene Gram-negative blood culture assay
7. Check KPC ESBL microarray
8. BioFire Film Array Method
9. Whole Genome Sequencing
10. Mass Spectroscopy  

Prospective Treatment Options for CRKP Infections

Since carbapenems are "the antibiotic of last resort," there aren't many active therapeutic choices for CRKP. There isn't a specific treatment plan in place, although high-dose antibiotics and a number of combination medicines are used as acceptable alternatives in clinical settings. Combination therapy is the most successful form of treatment because there is no recognised ideal course of action. the following are some typical therapeutics: 

1. Polymyxins (Colistin and Polymyxin B)
2. Tigecycline
3. Fosfomycin-tigecycline
4. Tigecycline-colistin-aminoglycoside  
5. Ceftolozane-tazobactam, 
6. Ceftazidime-avibactam, 
7. Imipenem-cilastatin-relebactam, 
8. Cefiderocol, 
9. Meropenem – Vaborbactam
10. A single dose of an aminoglycoside
11. Ampicillin – Sublactam (Cefoperazone – Sulbactam)
12. Carbapenem + aztreonam (in case of NDM, VIM and IMP producers)
13. Colistin + aminoglycosides/carbapenems/aztreonam
14. Rifampin + tigecycline/colistin 
15. Avibactam-ceftazidime
16. Plazomicin

Future/Alternative Treatment Options Under Study

1. Cefederocol 
2. Delafloxacin (Baxdale)
3. Nanoparticle therapy
4. Phage therapy 
5. Nucleic acid-based antibiotics 
6. Engineered endolysins
7. Vaccines 

Prevention and Control Measures from CRKP Infections

• measures for preventing and controlling infections, including good hygiene, sanitation, contact etiquette, and patient isolation
• Identification, isolation, and treatment of carriers 
• In hospitals, hand cleanliness is strictly enforced and required.
• regular monitoring of isolates for carbapenemase production and prompt communication of findings to the appropriate hospital officials
• maintaining the cleanliness of patient rooms and properly sterilising medical facilities 
• The strict use of appropriate and sufficient safety precautions by hospital employees 
• sterilisation of medical equipment before use 
• good surveillance system 
• preserving a sterile environment in the operating theatre, the operating room, the intensive care unit, and the ventilators  
• rationale for prescribing and using antibiotics
• The use of antibiotics requires a prescription. 
• Stewardship of antimicrobial treatments