Lecture #10: THE GRAM–NEGATIVE ENTERIC BACTERIAL PATHOGENS (GNEBPs)

 

THE GRAM–NEGATIVE ENTERIC BACTERIAL PATHOGENS (GNEBPs)

Gram-negative enteric bacterial pathogens (GNEBPs) are a group of organisms that reside mainly in the intestine and induce diarrhea. The bacteria included in this family share properties. They are Gram-negative, non–spore forming, straight rods (0.3–1.0 μm by 1.0–6.0 μm), motile by peritrichous flagella, or nonmotile. They grow well on peptone, meat extract, and usually MacConkey's media, both aerobically and anaerobically, often with gas production by fermentation, rather than oxidation. They are catalase-positive and oxidase-negative, reduce nitrate to nitrite, and have a 39–59% guanine and cytosine (G + C) content of deoxyribonucleic acid (DNA).

All members of the Enterobacteriaceae family ferment glucose with acid production and reduce nitrates. Enterobacteriaceae can cause a wide range of illnesses, which include wound infections, urinary tract infections, gastroenteritis, meningitis, pneumonia, septicemia, and hemolytic uremic syndrome.

These bacteria typically utilize oxygen for aerobic respiration, but when oxygen is absent such as in the gastrointestinal tract, they can perform fermentation.

Two Enteric organisms (Klebsiella pneumonia and Enterobacter species) are also included in the so called ESKAPE pathogen which are a group of antibiotic resistant bacteria that poses a threat to mankind in the coming decade.

The so–called SPICE organism are group of enteric that is unique because of its tendency to develop resistance to beta-lactam antibiotics. The genomes of SPICE organisms encode AmpC, an inducible beta-lactamase. The expression of this gene increases upon exposure to beta-lactam antibiotics, so organisms may initially appear susceptible and become resistant to beta-lactams within days or weeks. After stopping treatment, AmpC gene expression returns to baseline levels. Importantly, beta-lactamase inhibitors like clavulanic acid and tazobactam do not effectively inhibit AmpC beta-lactamases.

S	Serratia
P	Providencia
I	Indole–positive Proteus
C	Citrobacter
E	Enterobacter

The so called Carbapenem–Resistant Enterobacteriaceae (CRE) are a group of enteric that are resistant to certain antibiotics like carbapenem. Carbapenem antibiotics are generally considered to be the most potent group of antimicrobial agents with proven efficacy in the treatment of patients with severe bacterial infections, including those caused by otherwise antimicrobial-resistant strains.

SOME OF THE CARBAPANEM–RESISTANT BACTERIA
ENTEROBACTERIACEAE	NON–ENTEROBACTERIACEAE
Citrobacter freundii	Pseudomonas aeruginosa
Escherichia coli	Pseudomonas putida
Enterobacter aerogenes	Acinetobacter specie
Enterobacter cloacae
Enterobacter gergoviae
Klebsiella pneumonia
Klebsiella oxytoca
Proteus mirabilis
Salmonella enterica
Serratia marcescens

Lipopolysaccharides (LPS), as in other members of the family Enterobacteriaceae, consist of three domains: an endotoxic glycolipid (lipid A), an O-polysaccharide (O-PS) chain or O antigen, and an intervening core oligosaccharide (core-OS) region.

 

Classification of Enterobacteriaceae:

1.  Lactose fermenters (The Coliforms)

Coliforms are generally defined as Gram-negative, oxidase-negative bacteria capable of fermentation of lactose to acid and gas within 48 hours at 37°C. Many can survive below 5°C but without replication.

a.     Escherichia

b.     Citrobacter

c.      Klebsiella

d.     Enterobacter

2.  Non–lactose fermenters (NLF)

a.     Proteus

b.     Salmonella

c.      Shigella               

d.     Yersinia

e.     Morganella

f.      Providencia

3.  Delayed Lactose Fermenters (DLF)

a.     Serratia

b.     Edwardsiella

c.      Erwinia

d.     Hafnia

4.  Oxidase Positive

a.     Plesiomonas 

 

THE ESCHERICHIA

Escherichia is a gram-negative bacterium, which under the microscope is shaped like a rod with a small tail. It is composed of five species: Escherichia albetii, Escherichia coli, Escherichia fergusonii, Escherichia hermanii and Escherichia vulneris.

ESCHERICHIA COLI

Escherichia coli is part of the normal intestinal flora. Some strains are pathogenic and can cause gastroenteritis, UTI, meningitis, and wound infections. Some serotypes of Escherichia coli can produce toxins that result in blood-stained diarrhea or hemolytic-uremic syndrome. Vancomycin-resistant Escherichia coli (VRE) is one of the most common causes of infection in hospitals, accounting for up to 20% of all infections.

There are at least four Escherichia coli siderophores (enterobactin, aerobactin, salmochelin and yersiniabactin) with specific roles in iron uptake. Among these, yersiniabactin was shown to be the strongest predictor for extraintestinal virulence in a mouse model of sepsis but may also activate host responses such as autophagy in Crohn's Disease. Among these siderophores, it was found that salmochelin and yersiniabactin were produced more in UPEC strains.

Escherichelin, a byproduct of the yersiniabactin biosynthetic pathway in Escherichia coli, has been identified as an inhibitor of iron sequestration by Pseudomonas and its relatives.

ESCHERICHIA COLI NOMENCLATURE AND CHARACTERISTICS
PATHOTYPE	ACRONYM	CLINICAL PRESENTATION	TYPICAL GENETIC MARKER
DIARRHEAGENIC ESCHERICHIA COLI (DEC)
Enterotoxigenic Escherichia coli	ETEC	Mild to severe watery diarrhea, often in children in developing countries or travelers	Heat–labile enterotoxin (LT) and or heat–stable cholera toxin–like enterotoxin (ST), colonization factor (CFA/I, CS6, CS30)
Enteropathogenic Escherichia coli	EPEC	Attaching and effacing lesions on surfaces of intestinal epithelial cells, diarrhea, often accompanies by fever, vomiting and dehydration most often in children	Locus of enterocyte effacement (LEE) that includes intiminin, bundle– forming pilus
Enteroaggregative Escherichia coli	EAEC	Traveler’s diarrhea, often water, sometimes chronic, progression to HUS possible: aggregative adhesion with typical stacked–brick adherence pattern on Hep–2 cells	pAA virulence plasmids containing genes for aggregative adherence fimbriae
Verocytotoxin – producing Escherichia coli	VTEC	Attaching and effacing lesions, diverse pathotype: mild to bloody diarrhea (hemorrhagic colitis) accompanies by fever, abdominal cramping, or vomiting, can lead to hemolytic uremic syndrome (HUS)	Shiga toxin 1 or 2 (stx1 or stx2), LEE (in some but not all lineages)
Enterohaemorrhagic Escherichia coli	EHEC
Shiga toxin-producing enteroaggregative Escherichia coli	STEAEC
Enteroinvasive Escherichia coli	EIEC	Highly invasive; causes shigellosis/bacillary dysentery with profuse diarrhea, fever, and potential damage to intestinal walls; may proceed to HUS	Invasion plasmid pINV, Shiga toxin, enterotoxin on chromosomal pathogenicity island, lack of flagella
Diffusely adhering Escherichia coli	DAEC	Persistent watery diarrhea in children, diffuse adherence pattern, may be associated with Crohn’s disease and ulcerative colitis	Afa/ Dr adhesins
NON–DIARRHEAGENIC ESCHERICHIA COLI
Adherent invasive Escherichia coli	AIEC	associated with Crohn’s disease and ulcerative colitis	Lack of consistent virulence factors
EXTRAINTESTINAL PATHOGENIC ESCHERICHIA COLI (ExPEC)
Meningitis-associated Escherichia coli	MAEC	Older than 4 months have neck rigidity, tense fontanels, and fever. Older children and adults with may develop headache, vomiting, confusion, lethargy, seizures, and fever.	K1 capsule and pS88 antigen
Uropathogenic Escherichia coli	UPEC	Urinary tract infections (including recurrent), cystitis, pyelonephritis, prostatitis	PapGII and PapGIII
Avian Pathogenic Escherichia coli	APEC	Colibacillosis, septicemia, cellulitis in poultry
Sepsis–associated Escherichia coli	SEPEC	Bacteremia/sepsis
Unspecific ExPEC		Skin and soft tissue infections

Classification schemes applied to facilitate epidemiological studies and scientific discourse of Escherichia coli:

1.  First, serotyping is a classification method first developed in the 1970s based on properties of the 181 O-antigens (components of the surface lipopolysaccharide), 53 H-antigens (indicating the protein content of the bacterium's flagella, often encoded by the fliC gene) and the 80 capsule-based K-antigens.

2.  Second, MLST-derived sequence type (ST) describes the strain variability based on sequence information on seven housekeeping genes.

THE CITROBACTER

Citrobacter species are 1.0 × 2.0–6.0 μm in size. They are found either singly or in pairs, are devoid of a capsule, and are motile. Citrobacter species grow optimally at a temperature of 37°C. Its name suggests can utilize citrate as a sole carbon source. They grow on ordinary media as gray, opaque, round colonies that produce a strong, fetid odor. They also grow in the presence of potassium cyanide.

The genus Citrobacter was first proposed in 1932. Citrobacter contains 11 species: Citrobacter amalonaticus, Citrobacter farmeri, Citrobacter braakii, Citrobacter freundii, Citrobacter gillenii, Citrobacter murliniae, Citrobacter sedlakii, Citrobacter werkmanii, Citrobacter youngae, Citrobacter koseri, and Citrobacter rodentium.

Citrobacter species are not regarded as significant etiological agents in human disease. Citrobacter can cause septicemia in patients that display a number of predisposing factors. Citrobacter has also been found to cause meningitis, septicemia, and pulmonary infections in neonates and young children, and some of these cases have been linked to contaminated batches of PIF.

They are opportunistic pathogens in humans that can lead to invasive disease, including infections of the urinary tract, respiratory tract, CNS, skin, and soft tissue. The bacteria can cause osteomyelitis, suppurative arthritis, bacteremia, endocarditis, endophthalmitis, and intra-abdominal infections, particularly in neonates and immunocompromised hosts.

Citrobacter infections:

1.  Citrobacter freundii form small, circular, convex dark pink colonies on MacConkey agar. Rough or mucoid forms have also been reported. Citrobacter freundii and Citrobacter koseri can cause urinary tract infections, and are found in wounds, respiratory, meningitis, and sepsis. They can cause healthcare-associated infections, especially in pediatric and immunocompromised patients. They can resemble Salmonella on agar media due to production of H₂S by both genera. They are also citrate positive, however, only Citrobacter is positive for ONPG.

2.  Citrobacter koseri (formerly Citrobacter diversus) is uniquely associated with brain abscesses and capable of fermenting malonate.

3.  Citrobacter rodentium (formerly Citrobacter freundii strain 4280), is a nonmotile, gram-negative rod that ferments lactose but does not utilize citrate or does so marginally.

4.     Citrobacter werkmanii is an emerging and opportunistic human pathogen found in developing countries and is a causative agent of wounds, urinary tract, and blood infections.

THE KLEBSIELLA

The genus Klebsiella is a class of Gram-negative, encapsulated, nonmotile, rod-shaped, and oxidase-negative bacteria. They are facultative anaerobic Gram-negative straight rods (0.3 –1.0 μm in diameter and 0.6 – 6.0 μm in length). They grow best at temperatures between 12 and 43°C and are killed at 55°C in 30 minutes. They are capable of fixing nitrogen and are classified as associative nitrogen fixers or as diazotrophs. Several species produce bacteriocin, for example, klebosin.

Klebsiella species are chemoorganotrophic, having both a respiratory and a fermentative type of metabolism. They utilize glucose through the fermentation process originating acid or acid and gas, reduce nitrates to nitrites, and show negative oxidase and positive catalase reactions.

They are usually lysine decarboxylase positive and ornithine decarboxylase, arginine dihydrolase, and H₂S negative. Several species hydrolyze urea. Most species ferment all commonly tested carbohydrates, except dulcitol and erythritol; they also grow in the presence of KCN (potassium cyanide).

Two major subdivisions of Klebsiella are the Klebsiella pneumoniae species complex (KpSC) and the Klebsiella oxytoca species complex (KoSC). The KpSC currently includes five taxonomic species, two of which comprise two subspecies. These taxa were initially defined as seven distinct phylogroups, Kp1 to Kp7. In turn, the KoSC comprises 6 species and 7 phylogroups.

The clinically important species and subspecies are the Klebsiella pneumoniae, Klebsiella ozaenae, Klebsiella rhinoscleromatis and Klebsiella oxytoca.

The reservoirs for transmission of Klebsiella infections include the hands of health care workers and the gastrointestinal tracts of hospitalized infants.

THE KLEBSIELLA PNEUMONIAE SPECIES COMPLEX (KpSC)

The Klebsiella pneumoniae species complex (KpSC) has no formal taxonomic designation, and it commonly refers to closely related species that share 95%–96% average nucleotide identity with Klebsiella pneumoniae sensu stricto.

SPECIES OF KLEBSIELLA PNEUMONIAE COMPLEX
Species	Phylogroup
Klebsiella pneumoniae	Kp1
Klebsiella quasipneumoniae subspecie quasipneumoniae	Kp2
Klebsiella quasipneumoniae subspecie similipneumoniae	Kp4
Klebsiella variicola subspecie variicola	Kp3
Klebsiella variicola subspecie tropica	Kp5
Klebsiella quasivariicola	Kp6
Klebsiella africana	Kp7

KLEBSIELLA PNEUMONIAE

Klebsiella pneumoniae was classified as one of the ESKAPE organisms (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species), which are well-known highly virulent and antimicrobial resistant clinical pathogens.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) produces an enzyme known as carbepenemase that renders them resistant to carbapenem antibiotics.

ESBL stands for Extended Spectrum Beta-Lactamase. Beta-lactamases are enzymes produced by some bacteria that may make them resistant to some antibiotics like penicillins, cephalosporins, and monobactam aztreonam. ESBL-producing Klebsiella pneumoniae are resistant to those kinds of antibiotics.

Pathotypes of Klebsiella pneumoniae:

1.  Classical Klebsiella pneumoniae (cKP) – strains that have gained increasing notoriety owing to its propensity to accumulate mutations and acquire determinants, leading to the emergence of multiple, extensive, or pan-drug resistant clones.

2.  Hypervirulent Klebsiella pneumoniae (hvKp) – no specific and sensitive marker has been established to identify hvKp but several phenotypical and clinical features were defined for this variant. The hvKp strains rely on a battery of virulence factors for survival and infection, among which is the enhanced capsule production and the synthesis of siderophores.

a.  The first is their ability to cause severe infections associated with high pathogenicity and mortality in both immunocompromised and healthy hosts, typically presenting as pyogenic liver abscesses. Also, hvKp has been observed to trigger diseases at unusual sites, including endophthalmitis, necrotizing fasciitis, central nervous diseases (meningitis, bacteremia), etc.

b.  A second trait is their propensity for metastatic spread to distinct sites, which is uncommon among enteric Gram-negative bacilli, including cKP. Furthermore, a large proportion of hvKp colonies present a hypermucoviscous phenotype on agar plates which could be semi-quantitatively defined by a “string test.” The majority of reported hvKp clones belong to serotype K1 or K2.

THE KLEBSIELLA OXYTOCA SPECIES COMPLEX (KoSC)

The Klebsiella oxytoca complex is a human commensal but also an opportunistic pathogen causing various infections, such as antibiotic-associated hemorrhagic colitis (AAHC), urinary tract infection, and bacteremia, and has caused outbreaks.

They are non-spore forming and nonmotile and form smooth, circular, dome-shaped, glistening colonies on agar plates. Strains of all six named species of the Klebsiella oxytoca complex are positive for indole, lactose, lysine decarboxylase, mannitol, ONPG, and reduction of nitrate to nitrite but are negative for ornithine decarboxylase.

The positive indole test could differentiate species of the Klebsiella oxytoca complex from Klebsiella pneumoniae, while the positive ONPG test or the negative ornithine decarboxylase test could differentiate the complex from Raoultella ornithinolytica.

SPECIES OF KLEBSIELLA OXYTOCA COMPLEX
Species	Phylogroup	OXY variant(s)
Klebsiella michiganensis	Ko1	OXY–1, OXY–5
Klebsiella oxytoca	Ko2	OXY–2
Klebsiella spallanzanii	Ko3	OXY–3, OXY–9
Klebsiella pasteurii	Ko4	OXY–4
Klebsiella grimontii	Ko6	OXY–6
Klebsiella huaxiensis	Ko8	OXY–8
Taxon 1		OXY–10
Taxon 2		OXY–11
Taxon 3		OXY–12

KLEBSIELLA OXYTOCA

Klebsiella oxytoca is an important human pathogen causing a large variety of infections ranging from mild diarrhea to life-threatening bacteremia and meningitis. They colonize the skin, oral cavity, and intestinal and respiratory tracts of both healthy and sick people. Skin and soft tissue infections (SSTIs) due to Klebsiella oxytoca can be classified into three major types, i.e., wound infection, necrotizing fasciitis, and abscess.

Klebsiella oxytoca has been found in central nervous system (CNS) infection, endocarditis, endophthalmitis, septic arthritis, and many other types of infections, such as pleural empyema, prostatic infection, acute epididymitis, non–hemorrhagic diarrhea or colitis, and malignant external otitis.

Klebsiella oxytoca is a well-characterized causative agent of antibiotic-associated hemorrhagic colitis (AAHC), caused by the production of cytotoxins.

Toxins produced by Klebsiella oxytoca:

There are two distinct cytotoxins produced by Klebsiella oxytoca, tilimycin (also known as kleboxymycin or carbinolamine) and tilivalline (generated by nucleophilic attack of free indole on tilimycin which led to the pathological changes seen in AAHC).

Both tilivalline and tilimycin are pyrrolobenzodiazepine (PBD) metabolites and are generated from a bimodular nonribosomal peptide synthetase (NRPS) pathway.

1.  Tilimycin (TM) is a genotoxin interacting with double-stranded DNA, inducing cellular DNA damage in host cells in vitro and in vivo and causing a more serious lesion in cecal enterocytes.

2.  Tilivalline (TV) is not a protein but rather a pyrrolobenzodiazepine metabolite not previously linked with the intestinal microbiota and human disease. Indole enhances the conversion of tilimycin to tilivalline, an indole analog with reduced cytotoxicity.

KLEBSIELLA AEROGENES

Klebsiella aerogenes are oxidase-negative, rod-shaped bacterium that has peritrichous flagella that allows them to move. It is formerly known as Enterobacter aerogenes. They are isolated as human clinical specimens from respiratory, urinary, blood, or gastrointestinal tract. They are a nosocomial and pathogenic bacterium that causes opportunistic infections including most types of infections. They are salt-tolerant, able to produce gas and cause holes in the cheeses, leading to spongy bodies. They are also used to feed Naegleria fowleri and Acanthamoeba to multiply and differentiate into cyst in culture media.

THE ENTEROBACTER

Enterobacter is a genus of a common Gram-negative, facultative anaerobic, rod-shaped, non-spore-forming bacteria. They are commonly found in soil, water, and sewage. They also cause botanical disease. These organisms are facultatively anaerobic and motile by peritrichous flagella, except for Enterobacter asburiae. Most species yield positive results on malonate, citrate, sucrose fermentation, and Voges-Proskauer tests.

Enterobacter, Cronobacter, and Pantoea species are non-fastidious in nature and grow on blood and chocolate agar and selective media for enteric bacteria. On MacConkey agar, Enterobacter cloacae and Enterobacter aerogenes commonly appear as pink, lactose-fermenting, mucoid colonies similar in appearance to Klebsiella pneumoniae and Klebsiella oxytoca.

Three species of Enterobacter—Enterobacter cloacae, Enterobacter aerogenes, and Enterobacter sakazakii—are responsible for the vast majority of Enterobacter infections of humans. Pantoea agglomerans, formerly known as Enterobacter agglomerans, are also a common isolate and is grouped with the Enterobacter species.

ENTEROBACTER CLOACAE

Enterobacter cloacae is a well-known nosocomial pathogen contributing to bacteremia, endocarditis, septic arthritis, osteomyelitis, and skin/soft tissue infections, and lower respiratory tract- urinary tract and intra-abdominal infections. It also tends to contaminate various medical, intravenous, and other hospital devices.

The so called Carbapenem-resistant Enterobacter cloacae complex (CREC) are ECC strains which are resistant to antibiotics such as imipenem or meropenem.

The Enterobacter cloacae complex (ECC) are common nosocomial pathogens capable of producing a wide variety of infections, such as pneumonia, urinary tract infections, and septicemia.

ENTEROBACTER CLOACAE COMPLEX
Genovar	Specie
Cluster I	Enterobacter asburiae
Cluster II	Enterobacter kobei
Cluster III	unnamed Enterobacter cloacae Hoffmann clusters
Cluster IV	unnamed Enterobacter cloacae Hoffmann clusters
Cluster V	Enterobacter ludwigii
Cluster VI	Enterobacter hormaechei subspecie oharae
Cluster VII	Enterobacter hormaechei subspecie hormaechei
Cluster VIII	Enterobacter hormaechei subspecie steigerwaltii
Cluster IX	unnamed Enterobacter cloacae Hoffmann clusters
Cluster X	Enterobacter nimipressuralis
Cluster XI	Enterobacter cloacae subspecie cloacae
Cluster XI1	Enterobacter cloacae subspecie dissolvens
Cluster XI1I	Enterobacter cloacae sequence crowd

 

CRONOBACTER SAKAZAKII

Cronobacter sakazakii (previously known Enterobacter sakazakii) is a motile, peritrichous, Gram-negative rod that was previously referred to as a “yellow-pigmented Enterobacter cloacae.” This organism is ubiquitous in food products, being found in milk powder, rice, vegetables, cheese, sausage meat, teas, and various spices.

Cronobacter sakazakii can cause fatal invasive infection of neonates associated with the presence of this organism in powdered infant milk formula. The pathogen is also a rare cause of bacteremia and osteomyelitis in adults.

A new chromogenic medium (Druggan-Forsythe-Iversen agar) has been used to isolate Cronobacter sakazakii where they produce green colonies. Although the optimum temperature for their growth is 39°C, it is reported to grow at less than 4°C, suggesting that this species would be able to replicate even during refrigeration.

PANTOEA AGGLOMERANS

Pantoea agglomerans (formerly Enterobacter agglomerans), an anaerobic Gram-negative bacillus, is a rare cause of opportunistic infections. All species of the genus Pantoea can be isolated from feculent material, plants, and soil where they can be either pathogens or commensals.They are isolated in humans, resulting from soft tissue or bone/joint infections following penetrating trauma by vegetation.

Pantoea agglomerans bacteremia has also been described in association with the contamination of intravenous fluid, total parenteral nutrition, the anesthetic agent propofol, and blood products.

THE PROTEUS

The genus Proteus currently consists of five named species (Proteus mirabilis, Proteus penneri, Proteus vulgaris, Proteus myxofaciens, and Proteus hauseri) and three unnamed genomospecies (Proteus genomospecies 4, 5, and 6). They are Gram-negative, peritrichously flagellated rods capable of swarming growth on humid solid media. These bacteria are human opportunistic pathogens involved in many infections, but they mainly affect the urinary tract of hospitalized, long-term catheterized patients.

Biochemical Test	DIFFERENTIATION TEST AMONG
	Proteus	Providencia	Morganella
Citrate utilization	v	+	–
D–mannose fermentation	–	+	+
Gelatin liquefaction	+	–	–
H2S production	+	–	v
myo–inositol fermentation	–	v	–
Lipase (corn oil)	+	–	–
Ornithine decarboxylase	v	–	(+)
Swarming	+	–	–
Urea hydrolysis	+	v	+

Organisms belonging to the genera Proteus, Providencia, and Morganella are phylogenetically related members of the family Enterobacteriaceae and often referred to as the Proteeae. All members of these genera have phenylalanine deaminase activity, and most have urease activity and motility.

BIOCHEMICAL MARKERS FOR MEMBERS OF PROTEEAE
Organism	PAD	Mannose	ODC	Urease	Indole	Trehalose	Maltose	Adonitol	Xylose
Proteus mirabilis	+	–	+	+	–	+	–	–	+
Proteus vulgaris	+	–	–	+	+	v	+	–	+
Proteus penneri	+	–	–	+	–	v	+	–	+
Proteus myxofaciens	+	–	–	+	–	+	+	–	–
Morganella morganii	+	+	+	+	+	v	–	–	–
Providencia rettgeri	+	+	–	+	+	–	–	+	–
Providencia alcalifaciens	+	+	–	–	+	–	–	+	–
Providencia rustigianni	+	+	–	–	+	–	–	–	–
Providencia stuartii	+	+	–	–	+	+	–	–	–
Providencia heimbachae	+	+	–	–	–	–	v	+	–

In addition, the following properties are common to all members of the Proteeae: motility, inability to ferment dulcitol, lactose, sorbitol, raffinose and arabinose, lysine and arginine decarboxylase negative, malonate negative and mucate negative.

PROTEUS MIRABILIS

Proteus mirabilis has been implicated in bacteremia, neonatal meningoencephalitis, empyema, and osteomyelitis. This bacterium produces a cytoplasmic urea-induced multimeric, nickel metalloenzyme urease that hydrolyses urea to ammonia and carbon dioxide. The expression of this enzyme during UTI causes a substantial amount of ammonia to be produced.

Proteus mirabilis is positive for ornithine and negative for indole production, whereas Proteus vulgaris is negative for ornithine but positive for indole. Proteus penneri is a species that accommodates indole-negative, genetically distinct variants of Proteus vulgaris.

Proteus mirabilis and Proteus vulgaris are more accurately differentiated using the Ornithine Decarboxylase Test than by a test for Indole Production.

PROTEUS VULGARIS

Proteus vulgaris grows rapidly at room temperature and with penicillin grows in different forms. Moreover, it is a highly motile organism possessing many flagella and serves well for a study of the functions of these flagella. It is the only indole positive Proteus species.

It was previously considered biogroup 2 which has been reported to cause UTIs, wound infections, burn infections, bloodstream infections, and respiratory tract infections.

PROTEUS VULGARIS STRAIN BIOGROUP
Biogroup	Indole	Salicin Fermentation	Esculin Hydrolysis
1	–	–	–
2	+	+	+
3	+	–	–

Chondroitinase ABC (ChABC) is a bacterial enzyme from Proteus vulgaris. It is a lyase that degrades the chondroitin sulphate and dermatan sulphate chains of proteoglycan molecules; it also possesses hyaluronidase activity. It has been widely used as a strategy to promote repair following spinal cord injury (SCI). Most of the therapeutic effects of ChABC can be attributed to its ability to degrade the sugar chains from a class of proteoglycan molecules, the chondroitin sulphate proteoglycans (CSPGs).

PROTEUS PENNERI

Proteus penneri has been implicated in a case of bacteremia and concomitant subcutaneous thigh abscess in a neutropenic patient with acute lymphocytic leukemia and in nosocomial urosepsis in a diabetic patient from whom the organism was also subsequently isolated from bronchoalveolar lavage fluid and a pulmonary artery catheter tip. The urease enzyme of Proteus penneri is also believed to be a leading cause of kidney stone formation. Proteus penneri has also been isolated from stool and infected conjunctiva.

It is previously known as Biogroup 1 Proteus vulgaris which is characterized by negative reactions for indole production, salicin fermentation and esculin hydrolysis.

THE PROVIDENCIA

Providencia specie (named after the city of Providence, RI) are motile gram-negative bacilli that do not ferment lactose and are distinguished from other Enterobacteriaceae by their ability to deaminate phenylalanine and lysine.

They constitute the natural human gastrointestinal tract flora. It is commonly found in soil, water, and sewage. Human isolates of Providencia species have been recovered from urine, throat, perineum, axillae, stool, blood, and wound specimens.

Providencia species can be differentiated from Proteus species and Morganella morganii based on their ability to use citrate as the sole carbon source and to ferment D-mannitol.

Commonly, Providencia infections include urinary tract infections (UTIs), gastroenteritis, and septicemia. It is being increasingly reported from a plethora of other conditions like burns, pneumonia, neonatal sepsis, community, and hospital-acquired neuro infection, etc. Their isolation from clinical milieu is strongly associated with the presence of long-term indwelling urinary catheters in critically ill patients, diabetes, and other immunocompromised states.

Providencia species can deaminate aromatic amino acids including tryptophan and phenylalanine and can influence the formation of indole and indoxyl sulphate, which are metabolites of tryptophan. The production of indoxyl sulphatase or indoxyl phosphatase leads to the conversion of indoxyl sulphate into indigo and indirubin in the urine.

Providencia species are a gram-negative bacillus that produce bacterial urease, an important virulence factor associated with the formation of urinary tract stones, the obstruction of long-term urinary catheters, or the development of acute pyelonephritis.

The ability of Proteus rettgeri (now Providencia rettgeri) to produce acid from salicin, l-rhamnose, D-mannitol, adonitol, D-arabitol, erythritol, trehalose, and D-galactose formed to divide these strains into five biogroups together with their ability to hydrolyze or decompose urea.

DIFFERENTIATION TEST FOR PROVIDENCIA SPECIE
TEST	Providencia rettgeri	Providencia alcalifaciens	Providencia heimbachae	Providencia rustigianii	Providencia stuartii
Salicin	50	1	0	0	2
L– rhamnose	70	0	100	0	0
D–mannitol	100	2	0	0	10
D–adonitol	100	98	92	0	5
D–arabitol	100	0	92	0	0
D–galactose	100	0	100	100	100
Erythritol	75	0	0	0	0
i(myo) - inositol	90	1	46	0	95
Trehalose	0	2	0	0	98

PROVIDENCIA STUARTII

Providencia stuartii are urease-positive species, and urease activity is one of several factors which contribute to the development of urolithiasis. Specifically, Providencia stuartii and Proteus mirabilis co-infection contribute to the increased incidence of urolithiasis and bacteremia through synergistic induction of urease activity during co-infection.

PROVIDENCIA ALCALIFACIENS

Diarrheal infection caused by Providencia alcalifaciens usually presents as watery, non-bloody diarrhea, or loose stool, occasionally with abdominal pain, vomiting, fever, and rarely tenesmus.

Providencia alcalifaciens strains could produce the cytolethal distending toxin (CDT) which causes cell elongation, cell distention, and blocks eukaryotic cell proliferation at the G2/M phase, leading to cell death. It also uses manganese superoxide dismutase (Mn-SOD) for intra-phagocytic survival.

Providencia alcalifaciens medium (PAM) was found to be a suitable differential medium for Providencia alcalifaciens because it yields red colonies as opposed to yellow or white colonies observed in other Enterobacteriaceae.

PROVIDENCIA RUSTIGIANII

Providencia rustigianii produced acid from D-galactose but not from trehalose. Providencia stuartii produced acid from both while Providencia alcalifaciens produced acid from neither.

THE MORGANELLA

The genus Morganella currently consists of one species, Morganella morganii, with two subspecies, morganii and sibonii. It was positive in tests for indole production and the fermentation of carbohydrates but negative for the liquefaction of gelatin.

Morganella morganii subspecie sibonii contains three biogroups. Separation within biogroups E, F, and G is based on reactions with lysine and ornithine decarboxylases, production of indole, and growth in the presence of KCN.

Morganella morganii is an opportunistic secondary invader that was originally thought to be the cause of summer diarrhea.

Test	Morganella morganii subspecie morganii biogroup				Morganella morganii subspecie sibonii biogroup
	A	B	C	D	E	F	G
Indole production	100	100	100	100	100	75	56
Lysine decarboxylase	0	100	0	100	100	75	0
Ornithine decarboxylase	100	80	0	0	100	0	89
Growth in KCN	100	80	100	100	100	100	67
Glycerol	50	100	50	100	0	0	0
Rehalose	0	0	0	0	100	100	100

 

THE SALMONELLA

Salmonella is a group of Gram–negative, non–spore–forming prokaryotic rods. They are motile using multiple flagella but can switch to be non-motile in culture. They are facultative anaerobes and are catalase positive, oxidase negative and ferment glucose, mannitol, and sorbitol to produce acid or acid and gas.

Salmonella multiplies in reticuloendothelial cells and macrophages of the liver, spleen, lymph nodes, and bone marrow during the asymptomatic phase of infection. If left untreated, bacteria reach a threshold level and are released in the blood, initiating secondary bacteremia with cytokine secretion during the symptomatic phase of typhoid fever. This secondary persistent bacteremia phase leads to the seeding of other organs, which may further complicate the situation by developing intestinal perforations, hepatitis, pneumonia, and tissue abscesses, usually during the second and fourth weeks of illness.

The conventional scheme of serotyping of Salmonella evaluates the expression of surface antigens such as the thermostable polysaccharide cell wall or somatic (O), thermolabile flagella proteins or flagellar (H), and capsular (Vi) antigens. However, the Vi antigen is exclusively present in Salmonella typhi, Salmonella paratyphi C, and Salmonella dublin. It is also possible to subtype Salmonella serotypes based on phage typing.

There are only two species within the genus Salmonella: Salmonella enterica and Salmonella bongori. However, while only two species of Salmonella are recognized, their significance should not be underestimated given that Salmonella enterica itself consists of six subspecies with over 2500 serovars. Salmonella bongori can only infect cold-blooded animals.

Illness can range from mild to severe gastroenteritis and in some people invasive disease that can be fatal. Long-term sequelae such as reactive arthritis and irritable bowel syndrome have also been described as outcomes of salmonellosis.

Types of Salmonella infection:

1.  Non–typhoidal salmonellosis (NTS) refers to any illnesses caused to humans by all serotypes of Salmonella, except for the distinct typhoidal serotypes: Typhi and Paratyphi A-C. Salmonellosis is an acute, gastroenteritis, typically acquired orally through contaminated water or comestibles. The nontyphoidal serovar (NTS) Salmonella typhimurium can infect a wide range of hosts, including humans, plants, and animals.

It is among the most isolated foodborne pathogens associated with fresh fruits and vegetables such as apples, cantaloupes, alfalfa sprouts, mangos, lettuce, cilantro, tomatoes, melons, orange juice, celery, and parsley.

Salmonellosis is characterized by acute enterocolitis, which is accompanied by inflammatory diarrhea, a symptom rarely observed in individuals infected with invasive serovars (i.e. Salmonella typhi). Infection occurs after ingestion of >50,000 bacteria in contaminated food or water, with symptoms typically occurring 6–72 hours after consumption. Onset of symptoms is marked by abdominal pain and diarrhea with or without blood, while nausea and vomiting are also common. Typically, gastroenteritis will resolve itself in 5–7 days without need for treatment although symptoms are usually more severe and longer lasting in children.

2.  Invasive non–typhoidal Salmonella (iNTS) involves serotypes Salmonella typhimurium and Salmonella enteritidis; however, other serotypes such as Choleraesuis and Dublin are also known to cause invasive disease in humans. iNTS typically presents as a febrile systemic illness where diarrhea is often absent (as compared to non-invasive NTS salmonellosis and acute gastroenteritis, where diarrhea is common).

3.  Typhoid fever is caused by infection with Salmonella typhi. One difference between Salmonella typhi and NTS strains is the presence of the polysaccharide capsular antigen, Vi, which is thought to be a virulence factor of Salmonella typhi, allowing it to survive the acidic environment of the stomach early after infection, as acapsular Salmonella typhi is less virulent. Unlike NTS broad host specificity, Salmonella typhi is restricted to humans only.

Salmonella typhi can survive and replicate within host cells, particularly phagocytes (i.e. macrophages, dendritic cells, neutrophils, etc.), and the bacteria uses these cells to translocate to systemic sites of the body, such as the liver, spleen, and bone marrow.

SALMONELLA ENTERICA

Salmonella enterica (formerly Salmonella cholerasuis) is a Gram-negative food-borne pathogen causing enteric fever and gastroenteritis. It is rod-shaped, motile, facultative anaerobic pathogenic bacteria. The species enterica is further categorized into six subspecies: enterica, salamae, diarizonae, indica, arizonae, and houtenae, with about 2600 serovars based on surface antigens. Among all the serovars, the Salmonella enterica serovar typhi (Salmonella typhi) infection is strictly restricted to humans (known as typhoid fever). Based on somatic antigen, Salmonella enterica can be divided into 46 serogroups, and there are 114 individual flagellar antigens, resulting in 2600 serovars.

Only the Salmonella enterica subspecies enterica is of clinical relevance for humans and is further classified into more than 2,600 serovars. The human restricted serovar Typhi (STY) and the closely related serovar Paratyphi A (SPTA) cause enteric fever, while the generalist serovars Typhimurium (STM) and Enteritidis (SENT) are the most important causes of non-typhoidal salmonellosis.

DIFFERENTIAL CHARACTERISTICS OF SALMONELLA SPECIES & SUBSPECIES
Species	Salmonella enterica						Salmonella bongori
	enterica	salamae	arizonae	diarizonae	houtenae	indica
Dulcitol	+	+	–	–	–	d	+
ONPG (2hours)	–	–	+	–	–	d	+
Malonate	–	+	+	+	–	–	–
Gelatinase	–	+	+	+	+	+	–
Sorbitol	+	+	+	+	+	–	+
Growth with KCN	+	–	–	–	+	–	+
L(+) – tartrate	+	–	–	–	–	–	–
Galacturonate	–	+	–	+	+	+	+
γ-glutamyltransferase	+	+	–	+	+	+	+
ß-glucuronidase	d	d	–	+	–	d	–
Mucate	+	+	+	–	–	+	+
Salicine	–	–	–	–	+	–	–
Lactose	–	–	–	+	–	d	–
Lysed by phage O1	+	+	–	+	–	+	d
Usual habitat	Warm blooded animals		Cold blooded animals and environment

 

Laboratory identification:

1.  Xylose Lysine Deoxycholate agar (XLD) – will show a black center and a slightly red colored translucent zone due to the indicator color change. H₂S–negative Salmonella (e.g., Salmonella Paratyphi A) will grow pink with a dark pink center. Shigella will show red colonies as they do not ferment xylose, lactose, or sucrose.

2.  Hektoen Enteric (HE) agar – is a medium for the isolation of Shigella and Salmonella. It relies on the use of bile salts for selective inhibition and two indicator systems: (1) bromothymol blue and acid fuchsin as indicators of carbohydrate dissimilation and (2) ferric iron as an indicator of the formation of hydrogen sulfide from thiosulfate. Salmonella species produces transparent green or blue-green colonies with or without black centers and appears as almost completely black colonies. Shigella species produces green, transparent colonies.

3.  Bismuth Sulfite Agar is a selective and differential medium used for the isolation of Salmonella species from a variety of samples including foods and clinical specimens.  Salmonella typhi will appear as black colonies surrounded by brownish-black zones in the medium; a metallic sheen is normally present.

4.  The Felix–Widal Test (Widal Test) measures serum agglutinating antibody levels against the somatic O (LPS) and flagellar H antigens of Salmonella typhi bacteria. Two serum specimens, acute and convalescent, must be drawn from the patient at least 5 days apart. Like most serologic tests, a false-negative Widal test may occur early in the course of illness, and a false-positive Widal test may result from past infection or from previous exposure to cross-reactive antigens or vaccination. There are no universal standards that define the cutoff dilution of agglutinating antibodies to indicate a positive Widal test. The very low specificity of the assay (50% to 70%) and the inability to discern active from previous infection or vaccination means that the assay should rarely, if ever, be used.

 

THE SHIGELLA

All Shigella genera have no motility. They do not decarboxylate lysine. Neither citrate, malonate, nor sodium acetate can be used as the sole carbon source for the growth of Shigella (except Shigella flexneri). They cannot grow in the medium containing KCN and produce hydrogen sulfide. They are Gram-negative and facultative anaerobes. Man is the only known reservoir for Shigella specie.

Shigella and EIEC (Enteroinvasive Escherichia coli) share several common characteristics: for instance, EIEC are nonmotile, lysine decarboxylase negative, and often lack the ability to utilize lactose (although some EIEC and Shigella specie may be late lactose fermenters). Furthermore, EIEC share O-antigenic epitopes with some Shigella species and cause bacillary dysentery. However, EIEC strains tend to share more biochemical properties with Escherichia coli than Shigella, including the ability to produce mucate and acetate, indicating that they have retained chromosomally encoded genes, which have either been inactivated or lost from Shigella strains.

COMMON PHENOTYPIC MARKERS FOR THE DIFFERENTIATION OF SHIGELLA, EIEC & E. COLI
Phenotypic Marker	Shigella species	EIEC	Escherichia coli
Motility	–	–	+
Lysine decarboxylase	–	–	+
Sucrose	–	–	v
Xylose	–	+	+
Indole	–	+	+
PCR	+	+	–
Lactose	–	–/+	+
Christensen Citrate	–	–/+	+
Mucate	–	–/+	+
Acetate	–	–/+	+
Gas from glucose	–	+/–	+
Salicilin	–	v	v

Infections with Shigella species are often acquired by drinking water contaminated with human feces or by eating food washed with contaminated water. Transfer of shigella by flies has been very important during some outbreaks. They can be found in surface waters and within contaminated drinking water.

Shigellosis is also referred to as bacillary dysentery. The onset of symptoms typically begins 1–3 days following exposure but can range from 12 hours up to 6 days depending on the initial dose.

Virulence Factor:

1.  Shigella dysenteriae produces a potent exotoxin, Shiga toxin (Stx), which is only released upon lysis of the Shigella cell. Upon release, Shiga toxin binds to the surface of the host cell and is internalized by endocytosis. Once inside, the toxin halts host cell protein synthesis, thereby killing the host cell. Shiga toxin displays several toxic activities. It can act as an enterotoxin, a neurotoxin, and a general cytotoxin when given the opportunity.

2.  E-cadherin, a key protein involved in intercellular adhesion, has been shown to be an important cellular component involved in the intercellular spread of Shigella.

CLASSIFICATION OF SHIGELLA SUBGROUPS
SUBGROUP	SPECIES	SEROTYPES	FERMENTATION OF D–MANNITOL
A	Shigella dysenteriae	15	–
B	Shigella flexneri	8	+
C	Shigella boydii	19	+
D	Shigella sonnei	1	+

Laboratory isolation

1.  Salmonella Shigella Agar is a differential and selective medium for the isolation of Salmonella and Shigella species in pathological specimen and suspected foods. Salmonella species will appear colorless with black center while Shigella species will appear only colorless.

2.  HEX medium contains lactose, sucrose, d-xylose, and salicin as a differentiation marker. Shigella species produced green colonies on HEX medium, while Hafnia alvei, found to be false positive for Shigella on HE agar, appeared as differentiable orange colonies on HEX medium.

Please note that highly selective media like Salmonella Shigella Agar and Hektoen Enteric Agar are too stringent to promote growth of stress injured Shigella specie cells and are thus not recommended for the primary isolation of Shigella species.

THE YERSINIA

Yersinia are a group of Gram-negative, non-spore-forming, oxidase-negative, catalase-positive, lactose-negative, and facultative anaerobic rods (or coccobacilli). They can be transmitted by the consumption of contaminated food products including vegetables, milk products, and meat. The optimum growth temperature is about 30°C, and they, like Listeria monocytogenes, can grow at refrigeration temperatures. They can also survive the freezing process.

Yersinia strains are nonmotile at temperatures between 25 and 35°C. Yersinia enterocolitica and Yersinia pseudotuberculosis are psychotropic, being capable of growth at temperatures of 4 °C or even lower.

YERSINIA ENTEROCOLITICA

Yersinia enterocolitica is primarily a food-borne pathogen found in some food-producing animals such as pigs and other mammals. After ingestion of contaminated water or food, Yersinia enterocolitica colonizes the intestine causing yersiniosis, an acute gastrointestinal condition. Symptoms of yersiniosis include fever, abdominal pain, vomiting, and diarrhea. Treatment usually consists of an aggressive course of antimicrobial chemotherapy. Yersinia enterocolitica mainly causes acute gastroenteritis, but systemic infections, such as bacteremia, joint pain, and rashes have occasionally resulted.

Yersiniosis is often referred to as acute gastroenteritis. However, yersiniosis can have diverse symptoms that are not limited to the gastrointestinal tract. The course of yersiniosis is determined mainly by the age of the infected host.

BIOTYPES OF YERSINIA ENTEROCOLITICA AND THEIR PROPERTIES
BIOTYPE	SEROTYPE	PATHOGENIC PROPERTIES	SOURCE OF ISOLATION
1A	O:4, O:5, O:6,31, O:7,13, O:7.8, O:10, O:14, O:16, O:21, O:22, O:25, O:37, O:41, O:46, O:46, O:57, NT	Non–virulent or conditionally virulent	Soil, surface water, environment, game animals, vegetables, pigs, wild rodents, food producing animals
1B	O:4.32, O:8, O:13a, O:13b, O:16, O:18, O:20, O:21, O:25, O:41.42, NT	Highly virulent	pigs, contaminated vegetables, human feces, rodents, pork, milk products, refrigerated products
2	O:5.27, O:9, O:27	Weakly virulent	Pork products, milk products, asymptomatic pigs' tongues
3	O:1.2.3, O:3, O:5.27		Pork products, undercooked meat, contaminated vegetables, pigs, refrigerated products
4	O:3		Pork products, human feces, pigs, pets, milk products
5	O:3.2.3		Milk products, small ruminants, sheep, hares, pigs

YERSINIA PESTIS

Yersinia pestis is a nonmotile, nonsporulating, aerobic, Gram-negative bacillus or coccobacillus exhibiting a hairpin morphology after Gram staining and growing within 24 to 72 hours at a temperature range of 4 to 40°C (optimum, 28 to 30°C) at pH 7.4. It is microaerophilic, oxidase- and urease-negative, non–lactose-fermenting, and biochemically unreactive.

Yersinia pestis is the agent responsible for the plague which has been responsible for three human pandemics throughout history. These are the Justinian plagues, recorded from the sixth to eighth centuries, the Black Death from the fourteenth to nineteenth centuries, and the modern plague from the nineteenth century to the present time.

Biovars of Yersinia pestis

1.  Yersinia pestis subspecies pestis, considered typically human (divided into the Intermedium, Antiqua, Medievalis, and Orientalis biovars).

2.  Yersinia pestis subspecies microtus, considered typically zoonotic.

Types of Plague

1.  The most common initial presentation of plague is bubonic plague, which affects the lymph nodes and is characterized by the development of painful buboes (inflammatory swelling of the lymph nodes) in the groin, axilla, or cervical nodes. Symptoms initially include sudden onset of fever, chills, headaches, weakness, vomiting, and nausea, followed by the appearance of buboes (inflamed, tense, and painful due to replication of plague bacillus). The pain is often severe, leading to guarded movements and restricted mobility in the affected area. At advanced stages of the infection, the inflamed lymph nodes can turn into open sores filled with pus. The incubation period is no more than seven days after a person has been bitten by an infected flea.

2.  Septicemic plague is not clearly defined but is likely to occur within days of exposure. Septicemic plague can occur alone (absence of buboes) or secondarily to a bubonic form in combination with bubonic or pneumonic plague and results from a systemic infection with the bacteria. It can occur as a primary infection or can progress from bubonic or pneumonic plague. It involves the spread of the bacteria throughout the bloodstream, leading to septic shock and organ failure. Symptoms include fever, chills, weakness, abdominal pain, vomiting, diarrhea, and skin turning black and dying. It can be difficult to diagnose as it can present with non-specific symptoms that can be misinterpreted as sepsis. Septicemic plague is fulminant and lethal in the absence of rapid supportive therapy that includes effective antibiotic treatment. In instances of septicemic plague, individuals might present significant gastrointestinal symptoms, such as nausea, vomiting, diarrhea, and abdominal pain.

3.  Pneumonic or lung–based plague is the most virulent form and can occur when Yersinia pestis spreads to the lungs. Two clinical facts characterize this form, (1) a primary pneumonic plague, with 2 to 4 days of incubation following contact with a coughing patient, and (2) secondary pneumonic plague, which follows the dissemination of Yersinia pestis to the lungs during an episode of primary bubonic or septicemic plague. It can be transmitted via droplets from an infected person or animal. It is highly contagious, and person-to-person transmission can occur through the air. The incubation period for pneumonic plague is typically 1 to 3 days, and symptoms include high fever, chills, cough, chest pain, difficulty breathing (dyspnea), and possible expulsion of bloody sputum.

4.  Meningeal plague is the rarest form of plague. Plague meningitis follows the hematogenous seeding of bacilli into the meninges and is associated with symptoms of fever, headache, and meningismus and polymorphonuclear leukocytic pleocytosis.

5.  Pharyngeal plague would clinically appear to be viral or streptococcal pharyngitis, but the cervical lymphadenopathy associated with this would be much more painful and severe.

Forms of Plague

1.  The urban (domestic) form of plague, representing the epidemic manifestation, persists within rat populations and spreads among rats or between rats and humans through infected fleas.

2.  The sylvatic (wild) plague among small mammals, such as squirrels, prairie dogs, rabbits, and field rats, was first demonstrated in June 1916, in San Mateo County by gross anatomical examinations of squirrels (Citellus beecheyi). Although direct human contact with these rodents is rare, flea bites during such encounters can transmit the plague.

Laboratory identification:

1.  On MacConkey agar (48 hours) it grows as small, clear, or white non-lactose fermenter colonies.

2.  On Cefsulodin–Irgasan–Novobiocin (CIN) agar, the colonies are colorless, developing pink centers. Growth in broth culture at 35–37°C for 48 hours is flocculent, producing structures resembling “stalactite” and clumps at the side and bottom of tubes.

3.  Identified microscopically by examination of Gram, Wright, Giemsa, or Wayson’s-stained smears of peripheral blood, lymph node specimen, or sputum. The cells of Yersinia pestis appear as small pleomorphic Gram-negative rods (0.5–0.8 x 1–3 μm) by Gram staining.

4.  Xylose–Galactosidase agar (XGA) is a medium designed for recovery of Aeromonads, Salmonellae, Shigellae, and Yersiniae.

YERSINIA PSEUDOTUBERCULOSIS

Yersinia pseudotuberculosis is the least common of the three Yersinia pathogenic strains. It causes an illness characterized by fever and acute abdominal pain arising from mesenteric lymphadenitis, an inflammation of the lymph nodes.

There are 15 different O-serotypes (O:1–O:15) and 10 subtypes (O:1a–O:1c, O:2a–O:2c, O:4a–O:4b, O:5a–O:5b) based on variability in the lipopolysaccharide O-side chain (O-antigen).

On Cefsulodin–Irgasan–Novobiocin (CIN) agar, colonies of Yersinia pseudotuberculosis are smaller, deep red with a sharp border surrounded by a translucent zone.

THE SERRATIA

All species are Gram-negative straight rods, with rounded ends, 0.5 – 0.8 μm diameter and 0.9 – 2 μm in length. They are facultative anaerobes, catalase-positive and motile with peritrichous flagella. In a minimal medium containing ammonium sulphate as the nitrogen source, they can use many different compounds as sole carbon sources, including d-glucose, d-fructose, d-ribose, l-malate, l-aspartate, citrate, N-acetylglucosamine, gluconate and mannitol.

In liquid media, the cells are short rods known as swimmers, with one or two flagella. On 0.70 – 0.85% agar, they transform to swarmers – aseptate, elongated cells with 10 –100 lateral flagella. The flagellin proteins of swimmer and swarmer cells are identical.

Serratia is an opportunistic pathogen. It is normally found in the human intestine. It does not cause infections in otherwise healthy individuals.

Serratia produces two types of bacteriocins. Group A bacteriocin producing strains are resistant to chloroform, heat, proteolytic enzymes, and active against other Serratia strains. Group B bacteriocin producing strains are susceptible to these latter agents but are active against other enterobacteria but not against other Serratia strains.

Almost all species of Serratia have been isolated from foods, including fruits and vegetables. The Serratia hemolysin causes pore formation in erythrocyte membranes. This results in the osmotic lysis of erythrocytes, leading to the release of hemoglobin.

The genus Serratia includes Serratia marcescens, Serratia liquefaciens, Serratia rubidaea, Serratia odorifera, Serratia plymuthica, Serratia ficaria, Serratia entomophila, and Serratia fonticola.

Laboratory identification:

1.  Caprylate–Thallous (CT) Mineral Salts agar is a highly selective medium for the enrichment and isolation of all Serratia species. It contains 0.01% yeast extract, 0.1% caprylic (n-octanoic) acid as carbon source, and 0.025% thallous sulphate for inhibition of other organisms. Serratia colonies are small and slightly bluish-white.

2.  Tween 80 medium – contains 3.3% tryptose blood agar base, 0.4% Tween 80 and 0.015% CaCl₂. Serratia hydrolyses Tween 80 via an esterase, resulting in the release of free fatty acids, which in the presence of calcium form an opaque zone around the large, pinkish colonies.

SERRATIA MARCESCENS

Serratia marcescens is the species considered clinically important. It has been frequently encountered in hospital urinary or respiratory tract infections and in respiratory outbreaks and bacteremia outbreaks in day care centers and cardiac surgery and burn units. It is also associated with endocarditis, osteomyelitis, septicemia, wound infections, eye infections, and meningitis.

Three different types of bacteriocins, namely colicin L, mercescin A, B, and C, are produced by Serratia marcescens species. All are revealed as antimicrobial agents by inhibiting proteins, DNA and RNA syntheses, and the bacterial transportation process. Since it exhibits inhibitory activity against foodborne pathogenic bacteria, it can be applied to food preservation. An antimycotic metabolite, oocydin A, was isolated from epiphytic Serratia marcescens growing on an aquatic plant Rhyncholacis pedicillata.

Prodigiosin is a non-diffusible pigment produced by two biogroups (A1 and A2) of Serratia marcescens, and by most strains of Serratia plymuthica and Serratia rubidaea. Prodigiosin is best produced aerobically on peptone–glycerol agar at 12–36°C. Pyrimine is a water-soluble pigment that is produced by some strains of Serratia marcescens biogroup A4.

On blood agar and some other media, Serratia marcescens produces red colonies. As the colonies grow, the color intensifies.

SERRATIA ODORIFERA

There are two biogroups of Serratia odorifera which differed from each other by their ability to ferment two sugars and in their ability to decarboxylate ornithine.

1.  Biogroup 1 contains ornithine decarboxylase and can ferment raffinose and sucrose. Most of the isolates were cultured from the respiratory tract but were not pathogenic.

2.  Biogroup 2 cannot decarboxylate ornithine and is unable to ferment raffinose or sucrose. Most of the isolates were cultured from spinal fluid or blood, thus verifying that this biogroup can be pathogenic.

Serratia odorifera is a midgut inhabitant of Aedes aegypti mosquito that enhances its susceptibility to Dengue-2 Virus.

Bacterial contamination of red cells is most often due to Yersinia enterocolitica, followed by Serratia liquefaciens, whereas platelets are most often contaminated with Staphylococcus and Enterobacteriaceae.

THE EDWARDSIELLA

Edwardsiella is pathogenic to aquatic animals and occasional opportunistic pathogens for humans. They are known to cause gastroenteritis and wound infections and primarily isolated in stool samples.

Edwardsiella tarda is typically isolated from fresh or brackish water environments such as river mouths. It has also been isolated from the intestines of humans (after eating freshwater food sources such as catfish or eels) and from animals, including reptiles and freshwater fish. Approximately 80% of infections are intestinal. Edwardsiella tarda causes a Salmonella-like gastrointestinal infection, usually self-limited enteritis, with intermittent watery diarrhea and low-grade fever.

Edwardsiellosis, a bacterial septicemia caused by the Gram-negative bacterium Edwardsiella tarda, is a common but serious bacterial disease in cultured warm-water animals, including eels.

On Rimler–Shotts agar medium, it forms typical green colonies with black centers.

THE HAFNIA

Hafnia is not considered to be a major pathogen of humans, but rather it is an opportunistic pathogen generally affecting persons who are immunocompromised such as the elderly, the very young, and pregnant women as well as individuals with severe underlying diseases, such as hematologic malignancies, pulmonary diseases, cirrhosis/hepatitis, and pancreatitis.

The most common disease caused by the Hafnia species is bacteremia. It has also been associated with pneumonia, wound infections, urinary tract infections, cholecystitis, peritonitis, and arthritis.

Hafnia alvei (previously known as Enterobacter alvei) is the only named species of the genus Hafnia, although organisms presently classified as Hafnia alvei can be separated into three distinct genospecies.

In addition, Hafnia alvei appear similar to Escherichia coli O157 on sorbitol–MacConkey agar because both of these organisms commonly are D-sorbitol negative. H. alvei is lysine- and ornithine decarboxylase-positive and positive in the Voges–Proskauer test.

THE ERWINIA

Erwinia billingiae is a Gram-negative bacterial strain belonging to the Enterobacteriaceae family, first described in 1999 and has been associated with dermohypodermitis. Species in this genus are similar to Pantoea and Enterobacter species, and many Erwinia species have been reclassified into these groups.

THE PLESIOMONAS

The name Plesiomonas comes from the Greek word “neighbor,” and was chosen because the organism was believed to be closely related to Aeromonas when it is, in fact, more closely related to Proteus.

In the second edition of Bergey's Manual of Systematic Bacteriology, the genus Plesiomonas was officially transferred from the family Vibrionaceae to the family Enterobacteriaceae. The main reasons for this transfer were the following.

1.  Phylogenetic 5S, 16S, and multilocus sequence typing (MLST) data indicating that this taxon is rooted within the enterobacteria and not with the Vibrio and Photobacterium clades. 

2.  Plesiomonas shigelloides possesses a heteropolymer antigen linked to the lipopolysaccharide (LPS) termed the enterobacterial common antigen. This antigen is exclusively found in members of the Enterobacteriaceae and not in vibrios, including Grimontia hollisae and Photobacterium damselae.

3.  A number of cellular components, including polyamine composition and a Lipid A structure containing a common 1,4′-bis-phosphorylated-β1,6′-linked glucosamine backbone with six amide-linked acyl-oxyacyl groups is identical to that of Shigella sonnei.

PLESIOMONAS SHIGELLOIDES

Plesiomonas shigelloides is a motile, facultatively anaerobic, Gram-negative polarly flagellated rod native to aquatic animals and environments. It is the only oxidase–positive member of the family Enterobacteriaceae.

The positive oxidase, lysine, ornithine decarboxylase and arginine dihyrolase reactions and myo-inositol fermentation differentiates Plesiomonas shigelloides from other bacteria. A positive ornithine decarboxylase differentiates it from most Aeromonas species, and fermentation of myo-inositol differentiates it from both Aeromonas species and Vibrio species. It is also positive for indole and catalase.

The organism is most frequently found in subtropical or tropical climates though has a wide geographic distribution. After an incubation period of 1–2 days, patients typically develop watery diarrhea and vomiting, although some may develop dysentery. Diagnosis is via stool culture. Symptoms may last up to 2 weeks, although the disease is typically self-limited to immunocompetent individuals.

Plesiomonas enteritis can present in one of three forms: an acute secretory gastroenteritis (most common), bloody or dysenteric colitis, or chronic or persistent diarrhea of >14 days duration. There are additional reports of extraintestinal infections: meningitis in neonates, bacteremia, sepsis, and septic shock with high fatality rates. There is also stronger evidence that Plesiomonas diarrhea was associated with rotavirus coinfection rather than single infection. Extraintestinal infections, such as meningitis, bacteremia, and pseudoappendicitis have also been reported.

Laboratory identification:

1.  Polyphosphate inclusion bodies have been detected after 7 hours incubation in BHI broth that do not disappear during the stationary phase of growth, indicating that these bodies could be used as an energy source to initiate cell division under unfavorable environmental conditions. Being a fermentative and facultative anaerobic microorganism, Plesiomonas shigelloides can ferment glucose without gas production; all the strains ferment inositol and decarboxylate lysine and ornithine.

2.  For routine analysis of environmental and food samples for Plesiomonas shigelloides, spread plating on Inositol Brilliant Green Bile Salts (IBB) and plesiomonas (PL) agars is recommended. It also grows well on traditional enteric media, including MacConkey, Hektoen Enteric, Deoxycholate Citrate (Leifson), and Salmonella-Shigella agars. Growth is enhanced when inoculated on selective media, such as trypticase soy broth with ampicillin. They produce gray, non-hemolytic colonies after 18 – 24 hours of incubation at 37°C; optimal growth occurs at temperatures of 40°C – 44°C.