Lecture #13: NON-FERMENTATIVE GRAM NEGATIVE BACILLI

 

THE NON–FERMENTING GRAM–NEGATIVE BACILLI (NFGNB)

 

Non–fermenting Gram-negative bacteria (NFGNB) are a heterogenous group of Proteobacteria, which are characterized by their inability to ferment sugars to generate energy for their vital cellular functions. They are aerobic, non–spore-forming bacilli that either do not use carbohydrates as a source of energy or degrade them through metabolic pathways other than fermentation.

They occur as saprophytes in the environment, and some are also found as commensals in the human gut. Most often, NFGNB are niche pathogens that cause opportunistic infections in critically ill or immunocompromised patients.

They have been incriminated in infections, such as, septicemia, meningitis, pneumonia, urinary tract infections (UTI), and surgical site infections (SSI). NFGNB are innately resistant to many antibiotics and are known to produce extended spectrum ß-lactamases and metallo ß-lactamases.

 

THE PSEUDOMONAS

Pseudomonades are aerobic with respiratory metabolism where oxygen is the terminal electron acceptor; in some cases, nitrate can be used as a terminal electron acceptor and then growth occurs anaerobically.

They are motile by one or more polar flagella, and straight to curved rods that are 1.5–5.0 μm long with a 0.5–1.0 μm diameter. They are chemoorganotrophic and do not require organic growth factors. They are catalase positive and usually oxidase positive, are not very acid tolerant, and fail to grow below pH 4.5. They are vigorous, fast-swimming bacterium as seen in samples from the wild or from patients.

An earlier classification scheme grouped the pseudomonads into 5 homology groups based on similarities in 16S rRNA gene sequence. At present, only the former members of rRNA homology group I are retained in the amended genus Pseudomonas.

In 2010, Garcia–Valdez, et.al, made a partial sequence of four core “housekeeping” genes (16S rRNA, gyrB, rpoB and rpoD) of the type strains of 107 Pseudomonas species were analysed in order to obtain a comprehensive view regarding the phylogenetic relationships within the Pseudomonas genus.

Gene trees allowed the discrimination of two lineages or intrageneric groups (IG), called IG Pseudomonas aeruginosa and IG Pseudomonas fluorescens.

The first IG Pseudomonas aeruginosa, was divided into three main groups, represented by the species Pseudomonas aeruginosa, Pseudomonas stutzeri and Pseudomonas oleovorans.

The second IG was divided into six groups, represented by the species Pseudomonas fluorescens, Pseudomonas syringae, Pseudomonas lutea, Pseudomonas putida, Pseudomonas anguilliseptica and Pseudomonas straminea.

The Pseudomonas fluorescens group was the most complex and included nine subgroups, represented by the species Pseudomonas fluorescens, Pseudomonas gessardi, Pseudomonas fragi, Pseudomonas mandelii, Pseudomonas jesseni, Pseudomonas koreensis, Pseudomonas corrugata, Pseudomonas chlororaphis and Pseudomonas asplenii.

 

EARLIER CLASSIFICATION OF PSEUDOMONAS
Group	Palleroni Classification (1973)	Kersters Classification (1996)
I	Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, Pseudomonas mendocina, Pseudomonas alcaligenes, Pseudomonas pseudoalcaligenes
II	Pseudomonas mallei, Pseudomonas pseudomallei, Pseudomonas cepacia, Pseudomonas gladioli, Pseudomonas picketti	Burkholderia species, Ralstonia species, Cupriavidus species, Pandoraea species, and related organisms in the family Burkholderiaceae
III	Pseudomonas acidovorans, Pseudomonas testosterone, Pseudomonas delafieldi	Comamonas species, Delftia species, and Acidovorax species in the family Comamonadaceae;
IV	Pseudomonas diminuta, Pseudomonas vesiculare	Brevundimonas species in the family Caulobacteraceae
V	Pseudomonas maltophilia, Xanthomonas spp.	Stenotrophomonas species

PSEUDOMONAS AERUGINOSA

Characteristics:

1.     Pseudomonas aeruginosa is a Gram-negative, rod-shaped, asporogenous, motile, and monoflagellated bacterium. It has a pearlescent appearance and grape-like or tortilla-like odour.

2.     It is rod shape of 0.5–0.8 µm by 1.5–3.0 µm. The metabolism is oxygen-based respiratory, but this ubiquitous bacterium will grow in the absence of O₂ but in the presence of NO₃.

3.     It is an important opportunistic human pathogen, being a major cause of burn and eye infections and causing severe lung disease in cystic fibrosis patients.

4.     Pseudomonas aeruginosa needs a simple nutrition supply and can even grow in distilled water. It can also grow well in a medium containing acetate (carbon source) and ammonium sulphate (nitrogen source). The optimum temperature for growth is 37°C but can also grow at temperatures as high as 42°C. It is resistant to high concentrations of salts and dyes, weak antiseptics, and many commonly used antibiotics. Its ability to grow at 42°C helps distinguish it from many other Pseudomonas species.

5.     In addition to ETA and exoenzymes, P. aeruginosa secretes four main proteases; LasA (staphylolytic protease), LasB (elastase), alkaline protease, and protease IV.

6.     Pseudomonas aeruginosa strains produce two distinct types of O antigen (O-Ag): a common polysaccharide antigen (A-band) composed of a homopolymer of d-rhamnose, and an O-specific antigen (B-band) composed of a heteropolymer of three to five distinct sugars in its repeat units.

Laboratory Isolation:

1.     Cetrimide Agar is used as a selective medium for the isolation of Pseudomonas aeruginosa from pus, sputum and drains etc.

2.     Cetrimide Nalidixic Acid Agar is used for the isolation and enumeration of Pseudomonas aeruginosa from food and water samples.

3.     Acetamide Agar is recommended for differentiation of Pseudomonas aeruginosa from other non-fermentative gram-negative bacteria. Acetamide Agar can be used to test the ability of microorganisms such as Pseudomonas aeruginosa to utilize acetamide through deamination.

4.     King's A medium which uses magnesium and potassium salts to enhance production of the pigment pyocyanin.

EXTRACELLULAR PRODUCTS OF PSEUDOMONAS AERUGINOSA
Extracellular product	Empty Cell	Function
Proteases	Metalloproteases E	Breakdown of proteins, release of substrates
	Metalloproteases AP
	Elastase	Cleavage of transferrin: iron release
Phospholipase	Phospholipase C	Disruption of phospholipids in membranes and surfactants; hemolytic activity
Siderophores	Pyochelin	Growth in iron–limiting conditions
	Pyoverdin
ADP–ribosyl transferase toxins	Exotoxin A (ETA)	Inhibition of protein synthesis
	Exoenzyme S
Mucoid exopolysaccharide	Alginate	Adhesion, protection of cells from antibiotics, disinfectants, biofilm formation
Endotoxin	Lipopolysaccharide (LPS)	Major component of bacterial cell wall
Phenazine pigment pyocyanin		Strong reducing potential
Rhamnolipid		Biosurfactant: non-enzymatic hemolysin and cytolysin; strong reducing potential

Pigments produced by Pseudomonas aeruginosa:

1.     Pyoverdine (PVD), a siderophore produced by this bacterium, is essential for pathogenesis in mammalian infections. This observation is generally attributed to its roles in acquiring iron and/or regulating other virulence factors. Gallium nitrate has the ability to disrupt pyoverdine function.

a.     Siderophores are low-molecular weight compounds (<10 kDa) synthesized by microorganisms for iron sequestration and biocontrol of plant diseases. They comprise pyoverdins, catechols, hydroxamates, and rhizobactin. Siderophores fluoresce under UV light at 365 nm.

b.     Pyoverdine is a major class of siderophores synthesized by fluorescent strains of Pseudomonas putida, Pseudomonas syringae, and Pseudomonas aeruginosa.

c.      They are used by Pseudomonas aeruginosa to scavenge iron from the host proteins5, and acts as a signalling molecule for the production of two major virulence factors, exotoxin A and the endo-proteinase PrpL.

2.     Pyochelin (a derivative of pyocyanin) is a siderophore and can acquire iron from the host or in low-iron environments to maintain the pathogen growth.

3.     Pyocyanin contributes to the persistence of Pseudomonas aeruginosa in the lungs of Cystic Fibrosis patients, but also interferes with many mammalian cell functions, including cell respiration, ciliary beating, epidermal cell growth, calcium homeostasis and prostacyclin release from lung endothelial cells.

4.     Pyorubin is a nonfluorescent red pigment helping in protecting microorganism from oxidative stress.

5.     Pyomelanin, a polymer of homogentisic acid (HGA) is responsible for numerous biological properties and functions such as protection from light and oxidative stress, energy transduction as well as chelation and reduction of metal ions, e.g., Fe³⁺. Pyomelanin had been described as the result of the HGA autoxidation catalyzed by Mn²⁺ or Cu²⁺, from neutral to alkaline conditions.

Pathogenicity

Pseudomonas aeruginosa is pathogenic only when introduced into areas devoid of normal defense. This organism produces infection of wounds and burns giving rise to blue–green pus; meningitis, when introduced by lumbar puncture and urinary tract infection when introduced by catheter and instruments or in irrigating solution.

It also produces necrotizing pneumonia from contaminated respirators, often found in mild otitis externa in swimmers, may cause invasive (malignant) otitis externa in diabetic patients and may invade the bloodstream resulting in fatal sepsis in infants or debilitated patients.

Other pathogenic Pseudomonas:

1.     Pseudomonas putida, a specialized aerobic organism of the fluorescent group of Pseudomonas species, is a pathogenic bacterium of fish which can also colonize the human throat. It can be widely found in inanimate hospital surfaces and moist environments because of its strong tolerance to hard living conditions. They are reported in outbreaks from contaminated solutions such as distilled water, disinfectants, and transfusions, among others. They generally present as bacteraemia associated with endovascular devices.

A high proportion of Pseudomonas putida bacteraemia is catheter-related, occurring predominantly in immunocompromised hosts and generally associated with a low rate of mortality.

2.     Pseudomonas fluorescens is a commensal bacterium present at low level in the human digestive tract that has also been reported in many clinical samples (blood, urinary tract, skin, lung, etc.) and sometimes associated with acute opportunistic infections.

It has been isolated in respiratory samples from patients with lung transplants, ventilator-associated pneumonia (VAP), cystic fibrosis (CF) and rice-field drowning-associated pneumonia.

THE ACINETOBACTER

The name “Acinetobacter” originates from the Greek word “akinetos” meaning “unable to move,” as these bacteria are not motile, yet they display a twitching kind of motility presumably due to polar fimbriae. Bacteria of the genus Acinetobacter have gained increasing attention in recent years first, because of their potential to cause severe nosocomial infection, second, for their profundity in developing multidrug (MDR) and extreme drug resistance (XDR) third, for the ability of some strains to produce verotoxins (VA), and fourth, for the role members of the genus play in enhanced biological phosphorus removal in wastewater. Recently, Acinetobacter species have demonstrated a hydrocarbon-degrading capability, that is of interest for soil bioremediation and a specific strain Acinetobacter baylyi ADP1 has shown remarkable competence for natural transformation irrespective of DNA source, thus making it a potentially important tool for biotechnology.

Acinetobacter species are widely distributed in nature, in soil and water, as free-living saprophytes. They are found in virtually 100% of soil and fresh-water samples. They can colonize skin, wounds, and the respiratory and gastrointestinal tracts.

Acinetobacters are short, plump rods, typically measuring 1.0–1.5 × 1.5–2.5 μm when in the logarithmic phase of growth, but they often become more coccoid in the stationary phase. They are Gram-negative but may appear Gram variable, as is typical of members of the Moraxellaceae generally.

They are strictly aerobic, catalase-positive, indole-negative, oxidase-negative, non-fermentative encapsulated coccobacilli rods. Many strains are unable to reduce nitrates to nitrites.

Acinetobacter species also survive exposure to the commonly used disinfectants like chlorhexidine, gluconate, and phenols, particularly those not used in the appropriate concentrations.

PHENOTYPIC CHARACTERISTICS OF ACINETOBACTER SPECIES
Test	Abc complex	Acinetobacter lwoffii	Acinetobacter hemolyticus	Acinetobacter junii	Acinetobacter radioresistens
Catalase	+	+	+	+	+
Oxidase	–	–	–	–	–
Motility	–	–	–	–	–
Urease	v	v	–	–	–
Citrate	+	–	+	+	–
OF Glucose	+	–	v	–	–
Nitrate Reduction Test	–	–	–	–	–
Hemolysis	–	–	+	–	–
Gelatin hydrolysis	–	–	+	–	–
Growth at 42oC	+	–	–	–	–
Chloramphenicol sensitivity	R	S	R	R	R
Arginine hydrolysis	+	–	+	+	+

ACINETOBACTER BAUMANII

Acinetobacter baumannii is a Gram-negative bacillus that is aerobic, pleomorphic, and non-motile. An opportunistic pathogen, it has a high incidence among immunocompromised individuals, particularly those who have experienced a prolonged (> 90 d) hospital stay.

Acinetobacter baumannii specifically targets moist tissues such as mucous membranes or areas of the skin that are exposed, either through accident or injury. It can infiltrate open wounds, catheters, and ventilation tubes. It can cause fatal meningitis and pneumonia.

The Acinetobacter baumannii-calcoaceticus (Abc) complex is the most identified species in the genus Acinetobacter and it accounts for a large percentage of nosocomial infections, including bacteremia, pneumonia, and infections of the skin and urinary tract. It includes Acinetobacter baumannii, Acinetobacter nosocomialis, and Acinetobacter pittii.

The so called Carbapenem–Resistant Acinetobacter baumannii (CRAB) are bacteria that is resistant to nearly all antibiotics and difficult to remove from the environment. They carry antimicrobial resistance genes that allow the bacteria to make carbapenemase enzymes.

Laboratory Isolation:

1.     Blood Agar Plate (BAP) – colonies show typical morphology and size: non-pigmented, white, or cream colored, smooth or mucoid (when capsule is present).

2.     Eosin methylene blue agar (EMB) – colonies are bluish to bluish gray.

3.     Herellea agar (HA) – pale lavender in color.

4.     Leeds Acinetobacter Medium (LAM) – the colonies are pink on a purple background.

5.     Holton’s Agar – pink colonies with mauve background.

Virulence factor of Acinetobacter baumannii:

1.     Acinetobacter baumannii is known to produce and utilize three sets of siderophores: acinetobactins, fimsbactins, baumannoferrins. Acinetobactins and are chiefly made up of the amine histamine which results from histidine decarboxylation.  Siderophores are host iron-binding protein structures responsible for iron up take in bacteria. The role of acinetobactin as a virulence factor is associated with its ability to compete against the host iron sequestering proteins transferrin (Tf) and lactoferrin (Lf).

2.     The slime polysaccharides are toxic to neutrophils, and inhibit their migration as well as inhibit phagocytosis, but without disrupting the host immune system. They also confer the hydrophobicity of the bacteria.

3.     Outer membrane protein A (OmpA) binds to eukaryotic cells and gets translocated into the nucleus where it causes cell death.

4.     Other virulence conferring enzymes secreted by the bacteria include esterases, certain amino-peptidases, and acid phosphatases.

5.     Other key proteins that have been shown to contribute to Acinetobacter baumannii virulence include phospholipase D and C. While phospholipase D is important for resistance to human serum, epithelial cell evasion and pathogenesis, phospholipase C enhances toxicity to epithelial cells. Along with OmpA, fimbria, also expressed on the surface of the bacterial cell, contribute to the adhesion of the pathogen to host epithelia.

Other pathogenic Acinetobacter:

1.     Acinetobacter haemolyticus is widely distributed in nature and commonly found in soil, water, and hospital. Most of the strains causing infections are sensitive to a wide variety of antibiotics. The members of this clade show beta-haemolysis halos in blood-agar media, and sometimes they can also degrade gelatin.

2.     Acinetobacter radioresistens is a non–spore forming, aerobic gram-negative coccobacillus. Biochemically, it does not ferment lactose and is catalase positive and oxidase negative. It has previously been shown to cause septicemia in an immunocompromised individual where origin of septicemia was thought to be middle ear/sinus infection.

3.     Acinetobacter junii is a rare cause of disease, with documented cases of septicemia in neonates and pediatric oncology patients and a case of corneal perforation. It mainly affects patients who have had prior antimicrobial therapy, invasive procedures, or malignancy.

4.     Acinetobacter lwoffii (formerly Mima polymorpha) is a non-fermentative aerobic gram-negative bacillus that is seen as a normal flora of the oropharynx and skin in approximately 25% of the healthy individuals. Due to its ubiquitous nature, it is a potential opportunistic pathogen in patients with impaired immune systems, and it has been identified as a cause of nosocomial infections like septicemia, pneumonia, meningitis, urinary tract infections, skin, and wound infections.

5.     Acinetobacter colcoaceticus variety anitratum is formerly known as Herellea vaginicola.

BURKHOLDERIA PSEUDOMALLEI / PSEUDOMONAS PSEUDOMALLEI

Burkholderia pseudomallei, which causes melioidosis, is an aerobic, motile, non-spore-forming, gram-negative bacillus found in soil, vegetation, and water in tropical regions. Infection of the lung occurs more commonly because of spread through the bloodstream after cutaneous infection than as a result of inhalation.

Burkholderia pseudomallei, like many soil bacteria, is a difficult organism to kill. It can survive in triple-distilled water for years; it is resistant to complement and lysosomal defensins and cationic peptides; it survives inside several cell lines; and it produces proteases, lipase, lecithinase, catalase, peroxidase, superoxide dismutase, hemolysins, and siderophores.

The Burkholderia pseudomallei complex (BPC) now contains eight highly related species: Burkholderia pseudomallei, Burkholderia mallei, Burkholderia thailandensis, Burkholderia humptydooensis, Burkholderia oklahomensis, Burkholderia singularis, Burkholderia mayonis, and Burkholderia savannae.

Burkholderia pseudomallei infection:

Melioidosis is also referred to as Whitmore disease. It has been referred to as the “great mimicker” due to the wide variety of clinical symptoms that can be observed in patients presenting to hospitals around the globe. Pneumonia is the predominant clinical presentation of melioidosis and can appear in both acute and chronic cases, this is followed by non-healing skin lesions.

Melioidosis in humans is usually acquired by inoculation or inhalation. There is no evidence it can be acquired by ingestion. Occasional nosocomial infections have occurred, and sexual transmission has been reported twice. Rice farmers suffer repeated minor cuts and abrasions whilst immersed for much of the day in water containing Burkholderia pseudomallei, but they do not develop active infection unless an underlying predisposing condition develops.

BURKHOLDERIA MALLEI

Burkholderia mallei is the causative agent of both glanders (a naso-pulmonary syndrome) and farcy (cutaneous infection), which not only primarily infects solipeds such as horses and donkeys but can also cause fatal human disease upon exposure. Human cases are sporadic and most commonly associated with people working in proximity to infected animals.

Burkholderia mallei is a dangerous organism in the laboratory and should be investigated only in Biosafety Level (BSL) 3 facilities. It is highly infectious as an aerosol and, as infection requires only a few organisms, it has been used as a biological warfare agent.

BURKHOLDERIA CEPACIA COMPLEX (Bcc)

The Burkholderia cepacia complex (Bcc) is a group of opportunistic pathogens causing infections in patients with cystic fibrosis (CF), chronic granulomatous disease and other immunocompromised patients. They contain at least nine phylogenetically closely related yet distinct species, previously known as genovars.

Bacteria from the Burkholderia cepacia complex, which currently has over 25 different species, are transmissible among CF patients by social contact and intrinsically resistant to multiple antibiotics.

Bcc bacteria also possess the capacity to survive and proliferate in water-based environments, such as water bodies, lakes, rivers, drinking water, and liquids containing small amounts of nutrients. In addition to this capacity, these bacteria are described as major contaminants of sterile (e.g., intravenous drugs and solutions) and nonsterile pharmaceuticals (e.g., nasal sprays, water-based products, mouthwash, preoperative skin solutions, and hand sanitizers), being the cause of numerous nosocomial outbreaks registered.

The Burkholderia cepacia complex currently harbors nine genovars:

I.                Burkholderia cepacia

II.              Burkholderia multivorans, Burkholderia gladioli

III.            Burkholderia cenocepacia

IV.            Burkholderia stabilis

V.              Burkholderia vietnamensis

VI.            genovar VI

VII.         Burkholderia ambifaria

VIII.       Burkholderia anthina

IX.            Burkholderia pyrrocinia

Burkholderia cepacia, Burkholderia cenocepacia, Burkholderia vietnamiensis, Burkholderia anthina, and Burkholderia seminalis are commonly found in water-based environments worldwide.

STENOTROPHOMONAS MALTOPHILIA

Stenotrophomonas maltophilia, formerly named Pseudomonas maltophilia and then Xanthomonas maltophilia, is the only species in the genus. The name signifies “a unit feeding on few substrates,” based on the Greek roots stenos (narrow), trophos (one who feeds), and monas (a unit). Maltophilia means “affinity for malt,” based on the Greek roots maltum (malt) and philia (affinity).

Stenotrophomonas maltophilia bacteria are motile, free-living, glucose-non-fermentative, gram-negative aerobic bacilli with multitrichous polar flagella. They grow readily on most bacteriologic media, typically appearing pale-yellow, grayish, or lavender-green when grown on blood agar. Preliminary identification may be facilitated by its ammonia-like odor. Most clinical isolates are oxidase negative and uses maltose, dextrose, and xylose.

Stenotrophomonas maltophilia infections include pneumonia, acute exacerbations of chronic obstructive pulmonary disease, bacteremia, soft tissue and skin, cellulitis/myositis, osteomyelitis, catheter-related bacteremia/septicemia, meningitis, endophthalmis/keratitis/scleritis of the eye, dacryocystitis, endocarditis, UTI, biliary sepsis.

ALCALIGENES FAECALIS

Alcaligenes faecalis is a gram-negative rod with flagella. It is a nonfermentative aerobic, nonencapsulated, oxidase-positive bacterium and named for its ability to produce an alkaline reaction in certain medium.

Alcaligenes faecalis is commonly found in soil, water, environments as well as in human intestinal flora, and was first discovered in human feces. Nowadays, Alcaligenes faecalis is widely used in the sewage treatment, and several strains of Alcaligenes faecalis produce an important precursor, R-(—)-mandelic acid, essential for the production of various drugs and that is why, Alcaligenes faecalis is also widely used in pharmaceutical industries.

Additionally, in the environmental industries, some strains of Alcaligenes faecalis are used to degrade many organic contaminants in the biodegradation process of organic pollutants and industrial hazardous waste-materials.

Being a common bacterium in the human intestinal microbiota, Alcaligenes occasionally moves to blood and respiratory tract, and causes infections. Few rare cases of Alcaligenes faecalis-associated peritonitis, eye infections and urinary tract infections are available.

ELIZABETHKINGIA MENINGOSEPTICA

Elizabethkingia meningoseptica (formerly called Flavobacterium meningosepticum and, from 1994-2005, Chryseobacterium meningosepticum), is an environmental pathogen that is associated with opportunistic infection in humans. It is characterized by the formation of yellow to orange pigmented colonies on solid media. They also produce large, smooth colonies on blood and chocolate agar within 24 hours, but most isolates do not grow on MacConkey agar. Gram stain of agar isolates reveals long, thin rods that can be filamentous.

It is a nonfermenting, nonmotile, oxidase-positive Gram-negative aerobic bacillus that is ubiquitous in the environment, found in freshwater, saltwater, and soil.

Among the various serotypes of Elizabethkingia meningosepticum (A to F), type C has been the cause of most reported epidemics. As an agent of neonatal meningitis, it reportedly demonstrates mortality rates up to 57% and produces severe postinfectious sequelae including brain abscesses, hydrocephalus, deafness, and developmental delay.

Bacteremia is the second most common presentation of Elizabethkingia meningosepticum infection. It also causes endocarditis (including prosthetic valves), cellulitis, wound infections, sepsis following extensive burns, abdominal abscess, dialysis-associated peritonitis, and endophthalmitis. Infections including cellulitis, septic arthritis, community-acquired respiratory tract infection, keratitis, and bacteremia have been reported in the absence of underlying diseases.