Classes of Antibiotics:

Beta-lactam antibiotics are the most widely used class of drugs for the treatment of bacterial infections. They include penicillin and its derivatives, such as methicillin and amoxicillin. The beta-lactam ring portion of the antibiotic targets the penicillin-binding proteins (PBP), found in the bacterial cell membrane, which function in the synthesis of the cell wall. Binding of the antibiotic to the PBPs prevents the PBPs from performing their essential role and results in the death of the bacterial cell.

Gram positive bacteria acquire resistance to beta-lactam antibiotics through the production of a protein called PBP2a, which is able to avoid the inhibitory effects of the antibiotics. This is the mechanism by which MRSA is able to persist despite treatment with multiple beta-lactam antibiotics.

The genes for beta-lactamase enzymes are probably the most international in distribution: random mutations of the genes encoding the enzymes have given rise to modified catalysts with increasingly extended spectra of resistance. The archetypical plasmid-encoded beta-lactamase, TEM, has spawned a huge tribe of related enzyme families, providing ample proof this adaptability. The beta-lactamase genes are ancient and have been found in remote and desolate environments, which implied that novel beta-lactamases with altered substrate ranges occur in the environment. (Davies, “origins and Evolution of Antibiotic Resistance” Microbiology and Molecualr Biology Reviews, 2010, p. 417-433)

–Amoxicillin: is used to treat a wide variety of bacterial infections. It is a penicillin-type antibiotic that works by stopping the growth of bacterial. Amoxicillin is a widely utilized beta-lactam antimicrobial drug approved by the U.S. Food and Drug Administration (FDA) for use in the primary care setting. Amoxicillin is an aminopenicillin created by adding an extra amino group to penicillin to battle antibiotic resistance. This drug is indicated for the treatment of infections caused by susceptible isolates of selected bacteria, specifically those that are beta-lactamase–negative, including ear, nose, and throat infections, Helicobacter pylori eradication, lower respiratory and urinary tract infections, acute bacterial sinusitis, and skin and structure infections.

Amoxicillin is effective against a wide range of gram-positive bacteria, offering additional coverage against some gram-negative organisms compared to penicillin. Amoxicillin’s spectrum of activity includes coverage against Streptococcus species, with heightened efficacy against Listeria monocytogenes and Enterococcus spp. Furthermore, amoxicillin also demonstrates effectiveness against Haemophilus influenzae, select Escherichia coli strains, Actinomyces spp., Clostridium species, Salmonella spp., Shigella spp., and Corynebacteria spp.

Fluoroquinolones:

–Ciprofloxacin: is a member of the fluorquinolone drug class that is sued to treat various Gram-engative bacteria such as Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumoniae and E coli and Gram-positive bacteria such as Staphylococcus auereus. (Chegini, “Bacteriophages: The promising therapeutic approach for enhancing ciprofloxacin efficacy against bacterial infection” J Clin Lab Anal, 2023).

Gepotidacin: This antibiotic is in phase III (GSK). It could become the first novel oral antibotic treatment for uncomplicated urinary tract infections in over 20 years. Gepotidacin provides activity against most srains of E. coli, including isolates that are highly reistant to current antibiotics. It binds to two different enzymes, which would reuqire the bacteria to develop mutations in both enzymes to become resistant . GSK also ahs an exclusive licence with Spero Therapeutics for another antibiotic to treat complicated urinary tract infections called tebipenem pivoxil hydrobromide (tebipenem HBr). This drug belogs to a class of antibiotic agents called carbapenems, which are typically reserved for sever bacterial infections of suspecte mutlidrug-resistant bacterail infections.

Macrolide antibotics: such as erthyromycine were introduced to content with the problem of methicillin resistance and are widely used for the treatment of Gram-positive infections. The macrolides and related antibiotics act by binding at different sites in the peptide exit tunnel of the 50S ribosome subunit. Resistance can occur by modificaiton of the RNA or protein components of the tunnel.

Streptomycin: was introduced in 1944 for the treatment of tuberculosis. Mutant strains of Mycobacterium tuberculosis resistant to therapeutic concentrations of the antibiotic were found to arise during patient treatment.

Combination of antibiotics:

Antibiotic combination therapy is the application of two or more antibiotics and is widely sued in clinical settings to prevent the evolution of resistance. Compared to monotherpiees, such therapies can improve treatment efficiency, expand antibiotic coverage or reduce health damage ot humans. For example, beta-lactams are used in combination with aminoglycosides and fluoroquinolones for the treatment of gram-negative bacteria of sepsis and severe Pseudomonas infections.

Antibiotic adjuvants:

Antibiotic adjuvants can provide an alternative and complementary strategy that can target antibiotic resistance or enhcnace antibiotic action to restore or improve the antimicrobial activity of commonly used antibiotic. The adjuvants commonly used can be classified into beta-lactamase inhibitors, efflux pump inhibitors and outer membrane permeabilizers.

Antibiotic Resistance:

The gut microbioa in healthy adults is a reservoir for multiple ARGs. A major parte of the gut reistome has its origin in soil and water habitates, and some in food. As these populations had little exposure to antibiotics, their ubiquitous ARGs such as tetracylcine and several beta-lactam resistance genes are environmentally derived. Only a small fraction of ARGs pose a threat to human health. (Staley, “Long- and short-term effects of fecal microbiota transplantation on antibiotic reistance genes: results form a randomized placebo-controlled trial” (2024)

See also bioterrorism agents

Superbugs or bacterial strains which are resistant to antibiotics are becomming a major problem, particularly in hospitals. MRSA alone (below) causes more death each year than HIV/AIDS.

The majority of problematic infections with respect to failing antibiotic treatment is caused by the so called ESCAPE pathogens (acryonym slightly modified form “ESKAPE”) for Enteroccus faecium, S. aureus, Clostridium difficile, Acinetobacter-er baumannili, Pseudomonas aeroginosa and Enterobacteriaceae (incluced E. coli, Klebsiellia pneumoniae, Enterobacterspp). 

Enterrococcus faecium

Vancomycin Resistant Enterococcus becomes dangerous if it gets into your irinary tract or an open wound. VRE is a leading cause of urinary tract infections and meningitis. Mortality rate is about 40%.

S. Aureus (Methicillin Resistant Staphylococcus Aureus (MRSA) )

S. Aureus became a major threat in hospitals due to its high propensity for developing antibiotic resistance and ability to cause a multitude of different infections with high mortality. (Oleksewicz, Archives of Biochemistry and Biophysics 226 (2012) 124-131).

MRSA stands for methicillin resistant  (S. aureus) bacteria. MRSA was first recognized in 1961 about 2 years after the antibiotic methicillin was used to treat S. aureus. MRSA is a resistant strain of S. aureus exhibiting resistance against beta-lactamase penicillins, including methicillin.

The resistance to methicillin is due to a penicillin binding protein coded for by a mobile genetic element called the methicillin resistant gene (mecA). The gene has continued to evolve to the extent that many MRSA strains are currently resistant to several different antibiotics. S. aureus generally produces 4 types of cell wall composing proteins PBPS (penicillin bening proteins) (i.e., PBPS-1 to PBPS-4). However, MRCA produces a new type of PBPS called PBPS-2′. This type of PBP is a specific proteins which has poor affinity against beta-lactam antibiotics and is known to play a central role in the tolerance of this organism.

Signs and Symptoms:

MRSA infections of the skin tend to be raised, red, tender, localized lesions, often featuring pus and feeling hot to touch. They occur easily in breaks in the skin casued by injury, hsaving, or even just abrasion. They may localize around a hair follicle. Feveral is common.

Treansmission:

People become infected with MRSAS by physical contact with people who are infected or carriers (people not infected but who have the bacteria colonized on their body) or by physical contact with MRSAS on any object such as door handles, floors, sinks, or towels. People with a higher risk of MRSA infection include those with obvious skin breaks and those with depressed immune systems (infants, elderly, HIV-infected individuals).

Treatment

Treatment of MRSA often starts with incision of the lesion and drainage of the pus. Antimicrobial treatment should include more than one antibiotic. 

The Vancomycin group of antibiotics represent one of the last lines of defeinse against MRSA. These antiotics interfere with cell-wall biosyntehsis by binding to the D-Ala-D-Ala terminus of the disaccharyl pentapeptide of the peptidoglycan of the bacterial cell wall, resulting in cell death. Recently , however, even vancomycin resistant bacterial have appeared. These bacteria have been identified as having a D-Ala-D-lactate terminus rather than a D-Ala-D-ala terminus of the peptidoglycan. Vancomycin has a much lower affinity for the D-Ala-D-lactate terminus in vancomycin resistant bacterial and consequently makes it less effective as an antibiotic.

S. aureus is a prime target for anti-infective mAb tbased approaches. (Oleksewicz, Archives of Biochemistry and Biophysics 226 (2012) 124-131).

Detection: 

–coagulase test: Staphylococcal coagulase is an enzyme that reacts with factors in plasma to initiate clot formation. In the coagulase test, a tube of plasma is inoculated with the bacterium If it remains liquid, the test is engative. If the plasma develops a lump or becomes completely clotted, the test is postive. 

–genetic tests: The sequence of the mecA gene that codes for PBP-2′ is known. Thus hybridization methods employing a gene specific to mecA gene have been developed to detect MRSA.

Carbapenem-Resistant Bacteria

The emergence of gram-negative pathogens resistant to carbapenem antibiotics is one of the most pressing clinical problems worldwide, particularly when the pathogens produce β-lactamases that have full hydrolyzing activity against carbapenems. Since the description of the first carbapenemase, several of these enzymes, including Klebsiella pneumoniae carbapenemase (KPC), oxacilinase (OXA)-48–like carbapenemase, New Delhi metallo-β-lactamase (NDM), Verona integron–encoded metallo-β-lactamase (VIM), and imipenemase (IMP), have been disseminated globally and are commonly detected in Enterobacterales, Pseudomonas spp., and Acinetobacter spp. The Latin American Network for Antimicrobial Resistance Surveillance (ReLAVRA [Spanish acronym]. was established by the Pan American Health Organization/World Health Organization (PAHO/WHO) in 1996. Its goal is to inform AMR prevention and control policies and interventions in the region through ongoing collection of reliable, comparable, and reproducible AMR data focused on resistance surveillance of community-acquired and nosocomial pathogens. The network currently consists of 33 countries, 19 from Latin America and, since 2018, another 14 from the Caribbean; the organization was renamed ReLAVRA+ to note the additional countries. These countries are represented by their National Reference Laboratories (NRL), officially designated by the national authority in each country. Each NRL, head of the national network, is responsible for external quality control for all participating laboratories, routinely ensuring the reliability of the tests performed, including the validity of species identification and characterization, antibiotic susceptibility tests, and data quality. See Melano CDC

Resistant Acinetobacter Baumannii often occur in immunosuppressed patients such as those having invasive treatments in the hospital. It can cause severe central nervous system infections, meningitis and ventriculitis, especially in patients undergoing head trauma. It is also associated with urinary tract infections and pneumonia. Mortality rate is almost 80%.

Carbapenem-resistant Acinetobacter baumannii (CRAB), an opportunistic pathogen primarily associated with hospital-acquired infections, is an urgent public health threat.  In health care facilities, CRAB readily contaminates the patient care environment and health care providers’ hands, survives for extended periods on dry surfaces, and can be spread by asymptomatically colonized persons; these factors make CRAB outbreaks in acute care hospitals difficult to control.

Resistant E. coli 

Resistant E. coli s associated with gastrointestinal infections and dehydration. It comes from feces of an animal coming into contact with food products. It causes more than 3,000 death per year.

Clostridium Difficile 

Clostridum Difficile s passed in feces and spread to food and other objects when an infected person does not wash his/her hands. The bacteria generates spores which are very difficult to eliminate. It produces toxins that attack the lining of the intestine. Symptoms include diarrhea to inflammation of the colon, which can lead to death. Older adults in hosptials and long term facilities are particularly susceptible. Mortality is up to 25% in elderly persons.

C. difficile is a Gram-positive pathogen and the leading casue of antibiotic-associated diarrhea in the hspital setting. Nosocomial antibiotic assocaited diarrhea is a significant clinical problem with at elast 300 thousand cases eyarly in the US. Disease is caused by intestinal colonization with C. difficile and production of toxins A and B. Both toxins are cytotoxic (with toxin B being more poteint) and toxin A is also an enterotoxin.  (Oleksewicz, Archives of Biochemistry and Biophysics 226 (2012) 124-131).

Treatment

The standard of care is treatment with vancomycine or metronidazole, but relapse occurs in 10-20% of patients after cessation of treatment. and widespread vancomycin use may comproise its utility as a last resort drug.  (Oleksewicz, Archives of Biochemistry and Biophysics 226 (2012) 124-131).

Two human mAbs are currently in clinical development as adjuncts to treatment of antibiotic induced diarrhea in hospitalized patients agaisnt toxin A  and MDX-1388 agaisnt toxin B, developed by Massachusetts Biologic Laboratories/ Medarex/Merck. Both are fullyi human mAbs generated in transgenic mice using immunization with inactivated toxin A or inactivated toxin B combined with a recombinant protein correspoinding to the 590 C-terminal amiho acids of toxin B. (Oleksewicz, Archives of Biochemistry and Biophysics 226 (2012) 124-131).

Enterobacteriaceae

Klebsiellia Pneumoniae

–New Delhi metallobeta-lactamase (NDM-1) is rapidly spreading with infections appearing in both the UK and U.S. It is otherwise known as resistant Klebsiella Pneumonia and often associated with very difficult to treat blood stream infections, surgical site infections and meningitis. It was originally discovered in New Delhi. Mortality rate is over 50%.

Pseudomonas

Resistant Pseudomonas Aeruginosa: Pseudomonas is a type of bacteria (germ) that is found commonly in the environment, like in soil and in water. Of the many different types of Pseudomonas, the one that most often causes infections in humans is called Pseudomonas aeruginosa, which can cause infections in the blood, lungs (pneumonia), or other parts of the body after surgery. In 2017, multidrug-resistant Pseudomonas aeruginosa caused an estimated 32,600 infections among hospitalized patients and 2,700 estimated deaths in the United States. See CDC 

Psuedomonas Aeruginosa is a bacterium found in soil and water. It can get in through the skin and turn deadly. It is particularly serious in patients hospitalized with cancer, cystic fibrosis and burns. Mortality rate is about 50%. Pseudomonas aeruginosa is a common encapsulated, Gram-negative, rod-shaped bacterium that can cause disease in plants and animals, including humans. A species of considerable medical importance, P. aeruginosa is a multidrug resistant pathogen recognized for its ubiquity, its intrinsically advanced antibiotic resistance mechanisms, and its association with serious illnesses – hospital-acquired infections such as ventilator-associated pneumonia and various sepsis syndromes. 

Strains of Pseudomonas aeruginosa are known to utilize their high levels of intrinsic and acquired resistance mechanisms to counter most antibiotics. In addition, adaptive antibiotic resistance of P. aeruginosa is a recently characterized mechanism, which includes biofilm-mediated resistance and formation of multidrug-tolerant persister cells, and is responsible for recalcitrance and relapse of infections. The discovery and development of alternative therapeutic strategies that present novel avenues against P. aeruginosa infections are increasingly demanded and gaining more and more attention. See Cheng

Products of Pinesole have even beenr ecalled due to the present of Pseudomonas aeruginosa (see Consumer Product Safety Commission)

Curcumin as a flavonoid from the rhizome of Curcuma longa has antibacterial, antiviral and antifungal activity. Multidrug resistant non-fermenters are continuously increasing in hospital and ICU settings. One of the mechanisms of developing drug resistance is possession of efflux pump through which bacteria extrude antimicrobial agents and other toxic substances. If these efflux channels are blocked or inhibited, increased drug concentration can be achieved in a bacterial cell with optimal drug dose.  Significant reduction in MIC was observed after adding curcumin (50mg/L) with selected antimicrobial agents in 9/30 (30%) of multi drug resistant (MDR) isolates of Pseudomonas aeruginosa, while no change in MIC was observed when curcumin (50mg/L) was used alone, indicating its efflux pump inhibitor activity. See Mohapatra

Curcumin encapsulated in micellar/polymersome nanoparticles was also shown as an effective efflux pump inhibitor (EPI) on the expression of mexX and oprM genes in curcumin-treated and -untreated isolates of Pseudomonas aeruginosa. Clinical isolates of Pseudomonas aeruginosa were treated with ciprofloxacin (sub-MICs) alone and/or in combination with curcumin-encapsulated in micellar/polymersome nanoparticles. The expression of mexX and oprM genes was quantitatively evaluated by qRT-PCR in curcumin-treated and -untreated bacteria after 24 h. Curcumin-encapsulated in nanoparticles (400 µg/mL) induced cell death up to 50% in ciprofloxacin-treated (1/2MIC) resistant isolates during 24 h, while the bacteria treated with ciprofloxacin (without curcumin) were not inhibited. Also, curcumin in different concentrations increased effect of ciprofloxacin (sub-MICs). Downregulation of mexX and oprM genes was observed in cells treated with curcumin and ciprofloxacin compared to cells treated with ciprofloxacin alone. It seems that curcumin can be used as complementary drug in ciprofloxacin-resistant isolates through downregulating genes involved in efflux pumps and trapping ciprofloxacin on bacterial cells and increasing the effects of drug. See Takrami

Other Dangerous Bacteria

 Resistant Streptococcus is a strain that turns into a flesh eating disease. Mortality is about 28%. The fastest way to treat it is to cut the skin off.

The intestinal microbiota is confined most to the colon where some 1.5 kg of microbes or 1014 microorganisms reside. There are more genes in the human microbial than in the human genome. In the gut associated lymphoid tissue (GALT) enterocytes or intestinal epithelial cells represent the first barrier against invading organisms by secreting either mucin or defensins or sensing pathogens via TLRs. DCs also act as APCs. In aged GALT, a marked impairment of the immune response has been reported. Microbial components account for the production of short chain fatty acids (SCFA) such as butyrate, acetate and propionate. SCFA have antiinflammatory and anti-neoplasmic activities which exert a protective function. (Magrone, Immunity & Ageing 2013, 10: 31).

Bacteria living in the gut are being studies not only because they play a role in gastroinestinal disorders like inflammatory bowel disease, Crohn’s disease and colorectal cancer, but also because they can influence diverse and distal organs such as the brain. The gut-brain-axis begins with the enteric nervous system (ENS), a network of neruons that runs through the gastrointestinal tract. From there, the gut communicates with the brain via the vagus nerve, which connects the ENS to the central nervous system. 

Protection of Mucosal Surfaces

Protection of mucosal surfaces against conolinization and invasion by microbes is provided by a combination of constitutive, non-specific substances (e.g., mucus, lysozyme, lactoferrin and definsins) and also by specific immune recognition that relies on secretory IgA (SIgA). In the GI tract, microbe sensing and processing occurs in the Peyr’s patch tissue consisting of mucosal epithelium and associated lymphoid tissue. Microbes are sabled and ingestedc by the M Cell which passes them to APCs. (Corthesy, “Recombinant immunoglobulin A:powerful tools for fundamental and applied research” Trends in Biotechnology, 20(2), 2002, 65-71

Consequences of Disturbing Gut Microflora

Antibiotic-assocaited diarrhaea (AAD): Diarrhae is a frequent adverse effect of antiboiotic treatment and a common condition in hospitalised patients. The mechasmis by which antibiotics lead to AAD are multifaceted and include alteration of the normal intestinal microflora, often accompanied by overgrowth of patheogenic micro organisms. Nosocomial diarrhaea (NCD) is an entity defined as diarrhaea eveloped within 72 hf of hospitalisation (the 3 day rule). (Gorkiewicz, International J Antimicrobial Agents, 33(S1), 2009, S37-S41).

Synthetic Biotics:

Snthetic biotics invovles use of genetic engineering to create new biological parts. Synlogic for example first looks at underlying mechnisms or metabolic dysfunctions that cause a diase. This involves a review of the existing literature to identify and validate potential molecular targets. For example, it assed GI component and role of diet restriction when evaluating for example PKU and HCU as potential target indications. PKU is a rare genetic disease caused by the inability to metabolize or break down phenylalanine (Phe), an amino acid found in many foods, including vegetables, breads and creals. Over time, the toxic building of Phe can lead to severe neurological disorders and developmental delays in patients. HCU is caused by the loss of functional of the enzyme cystathione beta-synthase, which results in the toxic building of homocysteine and its metabolites in human blood and urine. Syptoms include life-threatening strokes caused by thromboembolism, lens dislocation, skeletal abnormalities and intellectual disability. Similar to that for PKI, HUC disease management involves adopting a highly restrictive diet (low in methionine, a precursor to homocysteine). Using molecular biology techniques it next tests prototypes that can carry out specific functions such as consuming toxins, regulating cellular funciton and targetting drivers of disease. Then standard molecular techniques to construct bacterial strains with key elements for devleopment such as the generation of auxotrophs, which are unable to synthesize one or more essential growth factors. The synthetic biotics are grown under controlling manufacturing conditions, lyophilized and formulated into a stable powder that can be adminsitered orally. These syntehtic biotics are desigend to be nonreplicating, noncolonizing and reversible by rapid GI clearance. Synlogic has made progress in development of the investigational synthetic biotics SYNB1618 and SYNB1934, which are engineered strains of E. coli Nissle encoding phenylaanine ammonial lyase (PAS), an enzyme that breaks down Phe. They are designed to metabolize Phe in the GI tract and convert it into a harmless metabolite. Synlogic is also developing the SYN81353 synthetic bioc for the potential treatment of HCU. It is an engineered strain of E. coli Nissie that consumes methionine in the GI tract ot prevent the accumulation of hemocysteine. That strain was developed as part of a research collaboration with Ginkgo Bioworks. 

Secretion of toxins is s a primary way bacteria cause disease.

Toxins are bacterial components that directly harm tissue or trigger destructive biologic activities. One common delivery mechanism of such toxins is the type III secretion system which is a pilli like apparatus that directly injects the toxin or effector molecules into the host cell which alter cell signaling. For example, enteropathogenic E coli uses a type III secretion system to deliver its own bacterially expressed receptor protein into the host cell which serves as a receptor for a bacterial adhesin which triggers a series of signalling events.  Y Pestis also uses a type III secretion mechanisms to get inside its host.

There are 2 types of toxins; endotoxins and exotoxins. Endotoxins are found only in  gram-positive bacteria. They are the lipid A portion of the lipopolysaccharide of the cell wall. Endotoxins bind to receptors on macrophages, B cells, etc and stimulate production of acute-phase cytokines, stimulate B cell growth. At a low concentration they result in fever, vasodilation and inflammation. At high concentration they can result in shock and death.

Exotoxins are produced by both gram-negative and  bacterial. They are often dimeric in structure with A and B subunits (A-B toxins). B binds to a cell receptor and A is transferred into the cell. Biochemical targets include ribosomes, transport mechanisms, intracellular signals. The genes for exotoxins are encoded on a plasmid or on a lysogenic phage. One important group of exotoxins are the superantigens. 

An example of an exotoxin is the Mycobacterium tuberculosis. Anthrax actually secretes 3 toxins,  I. edema factor, II. protective antigen and III. lethal factor. The Factor II (protective antigen) is responsible for getting the other 2 toxins into the cell.

Specific Types of Toxins

Cholesterol-dependent cytolysis (CDCs): are a large family of structurally related poreforming toxins which play various roles in the pathogenesis of a wide variety of human and animals diseases caused by Gram positive bacteria. A hallmark characteristic of the CDC is the dependence on membrane cholesterol for cytolysis. 

–Streptococcus intermedius intermedilysin (ILY): is a member of the CDCs. ILY is creted by Tereptococcus intermedius, a Gram positive pathogenic bacterium that can cause brain and liver abscesses. ILY is unusual among the CDCs in that it has been shown to specifically lyse human erythocytes; chimpanzee and cynomolgus monkey erythrocytes were 100 fold less sensitive to its cytolytic activity and all nonprimate erythrocytes examined to date are resistance its its cytolytic effects. Giddins (Natrual structural & molecular biology, November 2004) show that ILY uses humCD59 as a cellular receptor. The specificity of ILY for huCD59 is based on its recognition of the C8alpha and C9 binding domain of huCD59. Binding of ILY to human erythrocytes and ILY mediated lysis were also blocked by monoclonal antibody to huCD59. 

See also Antibiotic Resistance Mechanisms

In order to successfully colonize the gut and cause infection or disease, bacteria (or pathogenic bacteria) have evolved multiple virulence factors in interaction with the human microbiome that enable them to counteract colonization resistance.

Mobility and chemotaxis:

Flagellar motility and chemotaxis help bacteria to find sources of nutrients and localize suitable ecological niches for growth, improving the efficiency of bacterial environmental colonization. Falgellar mediated chemotaxis can assist bacteria to reach specific sites in new hosts, facilitating its colonization or invasion as well as growth and maintenance. (Yu, “The spread of antibiotic resistance to humans and potential protection strategies” Ecotoxicology and Environmental Safety 254 (2023).

Some studies have reported that essential oils and ethanolic extracts have been confirmed to reduce the invasion of Campylobacter jejuni on INT407 epithelial cells and motility through modulation of its LuxS system. Flagellar motility and its medaited chemotaxis are essential for bacterial colinization and thus the reduction of the flagellar motility of bacteria could b e a target for anti-colonization. (Yu, “The spread of antibiotic resistance to humans and potential protection strategies” Ecotoxicology and Environmental Safety 254 (2023)

Adhesion and invasion:

Adhesion and invasion are key steps in the colonization and infection of bacteria within the host. This procss requires the help of functional adhesins such as fibronectin-binding and laminin-binding proteins (Lmb). Fibronectin-binding proteins A and B is also important for persistent colonization of Staphylococcus aureus in the nose and intestine. Pili, especially tpe IV pili also play an important role in bacterial adhesion, ecological niche selction and establishment. Disruption of bacterial pilus-mediated adhesion processes can be achieved through inhibition of bacterail pilus production, adhesion inhibition, and adhesion based vaccines and antibodies. (Yu, “The spread of antibiotic resistance to humans and potential protection strategies” Ecotoxicology and Environmental Safety 254 (2023)

Type VI Section System:

The type VI secretion system (T6SS) is used in gram-negative bacteria and can deliver toxic substances to competitors and kill them or alter some of their key funcitons, which provides an advantage for the apthogen to conquer the colonization resistance of the host commensal bacterail. This promotes its colonizaiton and persistence. Effective inhibition of T6SS can reduce the invasion of commensal microbiome and niche dominance by exogenous pathogens. A mixture of organic acids and plant extracts was reproted to down-regulate the expression of T6SS related genes and reduce the virulence and cecum colonization of Camplobacter jenuni and Camplylobacter coli. (Yu, “The spread of antibiotic resistance to humans and potential protection strategies” Ecotoxicology and Environmental Safety 254 (2023)

Disruption of Human Gut Microbiome:

The human gut microbiome is well known to be an improtant reservoir of ARG and likely to be a key factor in regualting the emergency and spread of ARB. Antibiotic therapy for example can have a large impact on the human gut microbiome and its resistome, which eliminates not only pathogenic but also beneficial bacterial. The impact of antibiotics is influenced by the tyep of antibiotic, the route of adminsitration and the microbiome status of the patient. Antibiotics, expecially broad spectrum antibiotics, may generally have a negative impact on the diversity of the gut microbiome and promote the expansion of antibiotic specific ARG in humans. Appropritate routes of adminsitraiton combined with antibiotics with less impact on the gut microbiome can better reduce antibotic resistance while minimizing damage to the gut microbiome. (Yu, “The spread of antibiotic resistance to humans and potential protection strategies” Ecotoxicology and Environmental Safety 254 (2023)

Fecal microbiota transplantation (FMT), the delivery of feces form a healthy donor to the recipient’s intestine via enema or oral capsule, can rapidly reverse diseases assocaited with intestinal flora dysbiosis, enhance Cr, and limit increasing antibiotic resistance. FMT is theoretically the replacement of ARB in the recipient by a high abundance of non-antibiotic resistant bacteria.

Probiotics and prebiotic regulate the intestinal microbiota to enhance its CR intestinal barrier function. Some probiotic organisms (like lactobacillus rhamnosus GG, Lactobacillus casei Shirota, Bifidobacterium animalis Bb-12 and Saccharomyces crevisia boulardii have been reported to enhance nonspecific cellular immune response and miantain the gut homeostasis.

A healthy gut microbiome is critical for maintaining its CR and reducing exogenous bacterial colonization. Dietary fiber can promote the production of SCFA (including acetate, propionic acid and butyric acid) by relevant bacterial. SCFA plays an important role in maintaining the integrity of the gastrointestinal barrier and the loss of SCFA may lead to a decrease of CR against pathogenic bacteria. For instance, in patients receiving antimicrobila therpay, the abundance of the SCFA producing commensal bacteria decrased, accompanied by the expansion of carbopenem-resistant Enterobacteriaceae . SCFA inhibited the growth of antibiotic-restant E. coli and K. pneumoniae. Conversely, a high sugar, high fat, and high protein diet promoted colonizaiton by exogenous bacteria and promoted ARG expansion and transfer. (Yu, “The spread of antibiotic resistance to humans and potential protection strategies” Ecotoxicology and Environmental Safety 254 (2023)

Polyphenols like pomegranate ellagitannins, green tea polyphenols, rsveratrol, juice, blueberry and mango pup polyphenols and vitamins (A dn D) and Omega-3 fatty acids, also play an essential role in regualting microbiota and strenghtening barrier funciton. Polyphenols expand the population of beneficial species such as Bifidobacterium and Lactobacillus. and significantly enhance intesting related bacterial produciton of SCFA, which enhance intestinal barrier funciton.

Urease:

Urease is a virulence factor found in various pathogenic bacteria. It is essential in colonization of a host organism and in maintenance of bacterial cells in tissues. Due to its enzymatic activity, urease has a toxic effect on human cells. Urease is also an immunogenic protein and is recognized by antibodies present in human sera. The presence of such antibodies is connected with progress of several long-lasting diseases, like rheumatoid arthritis, atherosclerosis or urinary tract infections. (Kaca “Bacterial Urease and its Role in Long-Lasting Human Diseases” Current Protein and Peptide Science, 2012, 13, 789-806).

Ureolytic activity is often observed in pathogenic bacteria. Such a feature is characteristic of pathogenic Staphylococcus strains. Over 90% of clinical methicillin resistant Staphylococcus aureus strains are capable of urea hydrolysis. (Kaka “Bacterial urease and its role in long-lasting human diseases, Current protein Peptide Science, 2012).

In bacterial ureases, motives with a sequence and/or structure similar to human proteins may occur. This phenomenon, known as molecular mimicry, leads to the appearance of autoantibodies, which take part in host molecules destruction. Detection of antibodies-binding motives (epitopes) in bacterial proteins is a complex process. However, organic chemistry tools, such as synthetic peptide libraries, are helpful in both, epitope mapping as well as in serologic investigations. (Kaka “Bacterial urease and its role in long-lasting human diseases, Current protein Peptide Science, 2012).

Urease is capable of urea hydrolysis. This compound is widespread: it is found in the natural environment (water and soil) and in human body, where its occurrence is connected with protein degradation. In humans, urea is a factor of normal functions of kidneys. (Kaka “Bacterial urease and its role in long-lasting human diseases, Current protein Peptide Science, 2012).

A healthy adult excretes about 30 g of urea per day. However, it is present not only in urine, but also in blood serum, sweat and even in stomach.

Urease is a nickel-containing enzyme, which requires activity of a few additional proteins for acquisition of its hydrolytic properties. This process involves genes coding structural enzyme polypeptides as well as genes coding accessory proteins, located in a joint cluster.

The role of urease in bacterium surviving in unfavorable microenvironment in the host’s body is especially noticeable in case of H. pylori, a causative agent of gastritis and peptic ulceration. Ureolytic activity is essential for surviving M. tuberculosis, an etiologic factor of tuberculosis, a long-lasting inflammatory lung disease. Bacteria infect macrophages. They reside in phagosome, where alkalization due to ureolytic activity and subvert phagosome maturation takes place. Additionally, urease activity enables bacterium to exist in the environment where nitrogen sources are limited to urea.

See also metabolism

The process of bacterial metabolism begins with hydrolysis of large macromolecules in the external cellular environment by specific enzymes. Small molecules produced by this hydrolysis (i.e, fatty acids from lipids, monosaccharides from polysaccharides, short peptides from proteins) are transported into bacteria using a variety of transport mechanisms which all require permeases (binding proteins). These transport mechanisms include (1) facilitated diffusion (passive transport) down a concentration gradient (e.g., glycerol), (2) phosphorylation-linked transport where molecules are chemically altered during uptake (e.g., glucose) by phosphorylation, (3) active transport which requires a proton motive force (e.g, lactose) and (4) siderophores which traps Fe+3 from transferrin and transport it into the cell.

The process of substrate breakdown and conversion into usable energy is known as catabolism. The energy produced may then be used in the synthesis of cellular constituents (cell walls, proteins, nucleic acids, etc.) in a process called anabolism.  The specific  used by bacteria for catabolism and anabolism are, for the most part, shared by both prokaryotic and eukaryotic cells.

The primary pathway used by both bacteria and eukaryotic cells for the conversion of glucose to pyruvate (a universal intermediate) is the glycolytic or Embden-Myerhof-Parnas (EMP) pathway. The reactions which make up this pathway occur under both anaerobic and aerobic conditions. There is a net production of 2 molecules of ATP.

In the absence of oxygen, pyruvate undergoes fermentation wherein organic molecules are e acceptors and NAD is regenerated. Synthesis of key intermediates (anabolism) include malate, succinate, oxaloacetate, etc.

Under aerobic conditions, in the TCA cycle, pyruvate is oxidized to water and CO2 along with the generation of GTP and substrates such as alpha ketoglutarate, citrate, etc.. Additional ATP is also generated  in the electron transport chain from the oxidation of NADH which is provided by the TCA cycle.

Nitrogen Fixation of Some Bacteria:

Nitrogen fixation or the reduction of oxidized form of nitrogen into usable forms is the most energetically epxensive reaction to occur in any cell, requiring 16 ATP to make two molecules of NH3. This is due to the triple bond in N2.

Plants need ammonia (NH3) or nitrate (NO3-) to build amino acids, but most of the nitrogen in the atomosphere is in the form of gaseous nitrogen (N2). Plants lack the biochemical pathways (including the enzyme nitrogenase) necessary to cnvert N2 to NH3. Some bacteria have this capacity. Some nitrogen fixing bacteria such as Rhizbium fix nitrogen in exchange for carbohydrates from plants. Symboiotic relationships ahve evolved between some plant groups and bacteria that can fix atomospheric nitrogen into usable forms. Legumes for example can form root nodules which host hese bacteria in exchange for carbohydrates. This is important where the soil lacks nitrogen compounds.

Treponema pallidum, formerly known as Spirochaeta pallida, is a microaerophillic spirochaete bacterium with subspecies that cause the diseases syphillis, bejel (also known as endemic syphillis) and yaws, which are morphologically and serologically indistinguishable. It is transmitted only among humans. It is often described as Gram negative but its outer membran lacks LPS which is characteristic of other Gram negative bacteria. 

Lifecycle and Symptoms:

The clinical features of syphilis, yaws, and bejel occur in multiple stages that affect the skin. The skin lesions observed in the early stage last for weeks or months. The skin lesions are highly infectious, and the spirochetes in the lesions are transmitted by direct contact. The lesions regress as the immune response develops against T. pallidum. The latent stage that results can last a lifetime in many cases. In a few cases, the disease exits latency and enters a tertiary phase, in which destructive lesions of skin, bone, and cartilage ensue. Unlike yaws and bejels, syphilis in its tertiary stage often affects the heart, eyes, and nervous system, as well.

The etiological agent of syphilis is Treponema pallidum. Syphilis has diverse clinical manifestations and shares many clinical features with other treponemal and nontreponemal diseases. Therefore, it is mandatory that the clinical diagnosis is always supported by appropriate laboratory tests and that the test results are interpreted with reference to the patient’s history and physical examination findings. Syphilis progresses through distinct primary, secondary, latent and tertiary stages. The ulcers that appear in primary and secondary syphilis are rich in treponemes; venereal transmission occurs through direct contact with these lesions. The stage of the disease at which the patient presents has implications for diagnosis and treatment. In some stages, the disease may be asymptomatic, and there are problems in diagnosing very early syphilis, neurosyphilis, asymptomatic congenital syphilis and syphilis in intravenous drug users and persons coinfected with serologically cross-reacting agents and HIV.  See Ratnam “The laboratory diagnosis of syphilis”

Detection of T pallidum:

T pallidum, the etiological agent of syphillis, can not be seen using standard microscopic techniques. Instead, diagnosis is by darkfield microscopy or serology. 

In North America, many unsuspected cases are discovered by laboratory testing. The etiological agent, Treponema pallidum, cannot be cultured, and there is no single optimal alternative test. Serological testing is the most frequently used approach in the laboratory diagnosis of syphilis. Serological tests fall into two categories: nontreponemal tests for screening, and treponemal tests for confirmation. All nontreponemal tests measure both immunoglobulin (Ig) G and IgM antiphospholipid antibodies formed by the host in response to lipoidal material released by damaged host cells early in infection and lipid from the cell surfaces of the treponeme itself. All treponemal tests use T pallidum or its components as the antigen. If lesion exudate or tissue is available, direct examination is performed, followed by a nontreponemal serology test. A reactive nontreponemal test is then confirmed by a treponemal test. A confirmed serological test result is indicative of the presence of treponemal antibodies but does not indicate the stage of disease and, depending on the test, may not differentiate between past and current infection. Despite their shortcomings and the complexity of interpretation, serological tests are the mainstay in the diagnosis and follow-up of syphilis. Latent syphilis can only be diagnosed by serological tests. In fact, in North America, the majority of syphilis cases are identified at the latent stage by serological tests. The sensitivity and specificity of serological tests vary depending on the type of test and stage of the disease. See Ratnam “The laboratory diagnosis of syphilis”

One serological test is the Rapid Plasma Reagin (RPR) test, used to detect antilipid antibodies (reagin) present in the serum or plasma of persons with syphilis. Treponema pallidum, the etiological agent of syphilis, induces the production of at least two types of antibodies in human infection: anti-treponemal antibodies that can be detected by FTA-ABS antigen and anti-nontreponemal antibodies (reagin) that can be detected by RPR antigen.

For the test, the RPR antigen is mixed with unheated or heated serum on a plastic-coated card. If antibodies are present, they combine with the lipid particles of the antigen, causing them to agglutinate. The charcoal particles in an antigen suspension coagglutinate with the antibodies and show up as black clumps against the white card. If no antibodies are present, the test mixture is uniformly gray.  

Characteristic clumping ranging from marked and intense to slight from marked and intense to slight definitive clumping is Reactive. If the test is reactive, a semi-quantitative test to determine the highest titer giving a positive result. Titers are reported as 1:1, 1:2, 1:4, 1:8, 1:16, 1:32, etc. The higher the titer, the more antibodies present in the blood.

Treatment: 

T pallidum is sensitive to penicillin but more difficult to treat in patients with HIV infections.

Innate Responses to Lp

Upon entering macrophages, Lg replicate. But Macrophages mount response which induce pro-inflammatory cytokines (Il-12, IL-1 B, TNF, IL-1B, chemokines). Il-12 is important important because activates NK and NKT cells to secrete IFNy which activate macrophages which eliminate Lp.

Macrophages express TLRs which can recognize microbial patterns. Lp activates Macrophages most likely through TLR2.  (Hawn, Akamine, 2005, Girard) TLR2, but not TLR4, is an important molecule for host resistance against the intracellular growth of Lp as demonstrated by the ability of the bacterium to growth within macrophages from TLR2-/- mice, whereas TLR4 deficient macrophages did not affect Lp growth.

TLR5 has recently been reported to be repsonisble for the immune response to L p through the recongition of bacterial flagellin. (Hawn)

TLR9?

MyD88 is critical: K. Takeda and S. Akira, Cellular Microbiology 2003. All these TLRs (TLR5, 7, 9, 2, 4) signal through MyD88. Cytokine production by Macrophages in response to Lp requires MyD88. But Lp grows equally as well. So no inherent suspectibility ex vivo. However, in vivo, MyD88 is required for clearance of Lp.

Elimination of a single TLR is not sufficient to confer a Lp susceptibility phynotype as strong as that obserrved for MyD88 deficient m ice. In TLR2 knockout mice, there is a defect in IL-12p40. However, TLR2 deficient mice have a slight defect in clearance of Lp. No difference in clearance from the lung was observed for mice deficient in TLR4, TLR5, or TLR9.  So probably multiple TLRs are involved in response to Lp.

Innate Responses to Lp

If deplete CD4 or CD8 more suspectible to Lp. DCs are important for this adaptive response.  (Brieland, Engleberg, Friedman, Horwitz, Kline, Marre, Skerrett, Yamamoto.

Gordon D, Bray MA, Morley J. Prostaglandins produced by activated macrophages infected with Lp inhibit T cell responses.

Genetics of Host Resistance:

Chromosome 11 involved in suspeptibility to anthrax and chlamydiaie.  Gene on Chromosome 13 which confers resistence to Lp.

Colony forming unit (CFU): refers to individual colonies of bacteria, yeast or mold. A colony of bacteria or yeast refers to a mass of individual cells of the same organism, growing together. For molds, a colony is a group of hyphae (filaments) of the same mold growign together. CFUs are used as a measure of the number of microorganisms present in or on surface of a sample. To determine the number of CFUs, a sample is preapred and spread uniformaly on a surface of an agar plate and then incubated. The colonies that form are counted. 

CFU/mL=(# colonies)*dilution factor)/(volume plated in mL). So if you pipete 100 ul of the bacterial onto a plate with a 10 x 5 dilution, your CFU/mL = (400 * 10 to the 5th)(0.1 mL)=4*10 to the 8th) CFU/mL

Experiment; Determining CFUs (Legionella)

(1) spread your plates that have special agar for Lp growth on table. Do in triplicates for each sample.  Label the plates (time: eg. 24 hrs), (same #: eg., 1, 2…), (immature vs. mature DC, eg, i vs m), 

1 24 i LF only         2 24 i                1 24 PA only    2 24 PA only

1 24 i   LF only      2 24 i                1 24 PA only     2 24 PA only

1 24 i LF                2 24 i                1 24 PA only    2 24 PA only

(2) Stack plates under hood

1 24 10e1     1 24 PA only  ….

(3) Obtain polysterine glass tubes with plastic tops (kept in cabinet next to pcr). set up in groups as with plates in blue holder

(4) obtain 0.2% Saponin in water (lyses cells and releases bacterium from the cells). Use the small filter to refilter the saponin into a well plate

(5) add 100 ul to 100 ul of your samples. This will make [finial] saparin 0.1 %.   Time this for 10 min

Stop Reaction

(1) place HBSS into a 50 ml tube

(2) unscrew your polysterine tubes and place 800 ul of HBSS into each.

(3) after 10 min reaction time is up, transfer well of plate to polysterine tube (3 at a time, 100 ul)

(4) If need to you can do a dilution. Would put 800 ul of HBSS into 2nd set of polysterine tubes and take 100 ul of the 1st set and put into this set. This will be 10e2 dilution.

Streak Plates

(1) obtain bunsen burner (light one glass) and alcohol 95% ETOH.

(2) take sample polysterine tube, vortex and put 100 ul into plate

(3) light glass rod tip under burner, let cool slightly, steak.

(1) incubate plates for 3 -4 days to let bacterial grow.

(1) get w black agar plates (Jimmy makes it); (use 2 plates just in case 1 does not grow)  label LP on back of plate with Date and strain #. The vials are in last shelve of freezer (use Lp past through thioglycolated AJ Mqs. Swabs are in needles and swab drawer. 

(2) Dip sterile swab into vial (or pour entire contents of vial into plate if culturing only 24 hours. then use swab to spread it around on plate and use same swab to streak other plate) 

(3) streak (long) onto agar

(4) culture for 48 hours upside down (place in lower incubator).

(5) clean up area and use UV light for 10-15 min.