PFGE Typing Protocol Recommended by ARPAC for Acinetobacter baumannii

A genus of strictly aerobic, oxidase −ve, catalase+ve,Gram-type-negative bacteria of the family MORAXELLACEAE (within the
gamma subdivision of PROTEOBACTERIA); the organisms occur e.g. in soil and water and may act as opportunist pathogens in man.

Cells: short rods, 0.9–1.6 × 1.5–2.5 micron,or coccobacilli (coccoid in stationary-phase cultures);cells often in pairs. Nonmotile, but may exhibit TWITCHING MOTILITY. Non-pigmented.
Metabolism is respiratory (oxidative), with oxygen as terminal electron acceptor; no growth occurs anaerobically, with or without nitrate.
Most strains can grow on a mineral salts medium containing an organic carbon source such as acetate,ethanol or lactate as the sole source of carbon andenergy; some can use amino acids (e.g. L-leucine,ornithine) and/or pentoses (e.g. Larabinose,D-xylose), and some are able to degrade e.g. benzoate,n-hexadecane and alicyclic compounds.

Acinetobacters appear to contain all the enzymes of the TCA CYCLE and the glyoxylate cycle. Many carbohydrates can be used. Most strains in the A. calcoaceticus–A. baumannii complex (and in certain other groups) can form acid from glucose (oxidatively), but many (e.g. most strains designated A. lwoffii ) cannot. The optimal growth temperature is typically 33–35°C.

GC%: ∼38–47. Type species: A. calcoaceticus.

The taxonomy of Acinetobacter is confused and unsatisfactory.Emended descriptions of the two species A. calcoaceticus and A. lwoffii, and proposals for four new species (A. baumannii, A. haemolyticus, A. johnsonii and A. junii ), were published in 1986 [IJSB (1986) 36 228–240]. Since then, a number of adjustments have been made to the taxonomic structure of the genus.

Acinetobacters have been isolated in a number of hospitalassociated (and other) outbreaks of disease, often as part of a mixed infection; in most cases such infections involve glucolytic strains of the A. calcoaceticus–A. baumannii complex– particularly A. baumannii
(also called group 2, or genospecies 2).

The most common manifestations of disease include septicaemia and infections of the urinary tract, lower respiratory tract and central nervous system. Transmission may occur by direct contact or may involve the airborne route.

Acinetobacters have been reported to survive on dry surfaces for at least as long as e.g. Staphylococcus aureus.

One problem associated with the pathogenic role of Acinetobacter is that these organisms appear easily to acquire resistance to antibiotics – so that they have the potential to develop as multiresistant pathogens.

Protocol ARPAC

PFGE method

Organism--------Acinetobacter baumannii

Recommended lysis enzyme:proteinase K

Restriction enzyme---------SmaI, ApaI

Approximate no. of---------20–40 , 20–30
restriction Fragments

Fragments size------------5–300,10–300
range (kb)


A.baumannii ACICU:

Methods for Typing Proteus spp.

Proteus mirabilis HI4320( Genome)

Proteus mirabilis HI4320, complete genome

Protein coding

"Complete genome sequence of uropathogenic Proteus mirabilis,a master of both adherence and motility."

Sanger: Genome Proteus mirabilis strain HI4320

PFGE method

Organism-------------------Proteus mirabilis

Recommended lysis enzyme:proteinase K

Restriction enzyme---------SfiI,NotI

Approximate no. of---------7–10,6–10
restriction Fragments

Fragments size------------50–700,75–700
range (kb)

General Information

Proteus spp.


Characteristics of Proteus Species.

The genus Proteus includes five species, the most common of which are P. mirabilis and P. vulgaris. Proteus species are commonly found in the environment and as normal flora in the intestinal tract of humans and other animals.

Proteus mirabilis, like other members of the Enterobacteriaceae family, is a nonspore-forming, facultative anaerobic, gram-negative bacillus.
Proteus has fimbriae, which facilitate attachment to uroepithelium, and flagellae,which provide the motility required for ascending infection.

Proteus also has the ability to transform from a single cell form to a multicell elongated (swarmer) form. The swarmer cells are more likely to be associated with cellular adherence in the kidney as demonstrated in an animal model of infection.
Hemolysin, which induces cell damage by forming pores, may also play a role in establishment of pyelonephritis.

Diagnosis of a UTI is initially by the urinalysis followed by a culture.


Proteus is easily identified on a MacConkey agar plate as a clear colony (nonlactose fermenter). The obvious swarm seen on blood agar would indicate a Proteus species.

P. mirabilis and P. vulgaris would be indole positivity in the latter.
Proteus species are usually among the most susceptible genera of all of the Enterobacteriaceae, most susceptible to penicillin, although it is not uncommon for them to be resistant to tetracyclines.
Proteus vulgaris, however, tends to be resistant to more antimicrobials than P. mirabilis.

Proteus species produce infections in humans only when the bacteria leave the intestinal tract. They are found in UTIs and produce bacteremia, pneumonia, and focal lesions in debilitated
patients or those receiving intravenous infusions.

Proteus mirabilis is a common cause of UTI. Proteus species produce urease, making urine alkaline and promoting stone formation. The rapid motility of these organisms is evidenced by “swarming” a thin film of organisms over the entire agar plate.
Proteus species produce a urease, which hydrolyzes urealeading to ammonia, which alkalinizes the urine (leading to a higher pH).
Both proteus and E. coli are gram-negative rod bacilli.

Methods for Typing Enterococcus spp.

Role of Mobile DNA in the Evolution of Vancomycin-Resistant Enterococcus faecalis (Genome)

Enterococcus faecalis V583 Genome

Genome Properties Report


Genes assigned a role category

Organism Gene List

PFGE method

Organism-------------------Enterococcus spp.

Recommended lysis
enzyme:lysostaphin,lysozyme,proteinase K

Restriction enzyme---------SmaI

Approximate no. of---------15–20
restriction Fragments

Fragments size------------5–400
range (kb)

General information

Enterococcus

Methods for Typing klebsiella spp.

Complete Genome Sequence Klebsiella pneumoniae 342

Klebsiella pneumoniae Kp342 Genome

Genome Properties Report

Genes assigned a role category

Organism Gene List

PFGE method

Organism-------------------Klebsiella spp.

Recommended lysis enzyme--proteinase K

Restriction enzyme---------XbaI

Approximate no. of---------15–20
restriction Fragments

Fragments size------------10–700
range (kb)

General information

The genus Klebsiella, which belongs to the includes family Enterobacteriaceae five species,with the most clinically significant being K. pneumoniae.

Klebsiella pneumoniae is a large, nonmotile, with a prominent polysaccharide gram-negative rod capsule.
The capsule is antiphagocytic and retards leukocyte migration into an infected area.

Another virulence factor of K. pneumoniae is its propensity to develop resistance to multiple antibiotics.All strains of K. pneumoniae are innately resistant to ampicillin, because of the production of β-lactamase.

Acquisition of resistance to other antibiotics usually occurs by transfer of plasmids from other organisms.Recently strains of nosocomially acquired K.pneumoniae have been isolated that produce an extended spectrum β-lactamase and therefore are resistant to all β-lactam antibiotics.

Klebsiella pneumoniae will grow rapidly producing large mucoid colonies on routine laboratory media.Colonies are often extremely mucoid and will tend to drip into the lid of the plate while incubating in an inverted position. Pink colonies will be evident on MacConkey agar indicating their fermentation of lactose.
Both K. oxytoca and especially K. rhinoscleromatis are slower growing than
K. pneumoniae and the other Enterobacteriaceae. All Klebsiella species are very closely related with nearly identical biochemical reactions, except for the fact that K. pneumoniae is indole negative, and K. oxytoca is indole positive.

Treatment of K. pneumoniae pneumonia would be based on the susceptibility of the isolate. Treatment can be complicated by the presence of multidrug-resistant strains.Most strains are susceptible to extended spectrum cephalosporins such as cefepime as well as fluoroquinolones such as moxifloxacin. In cases of strains that produce an extended spectrum β-lactamase, the treatment of choice would be imipenem or meropenem.

Prevention of spread in the hospital would involve appropriate infection control procedures to isolate patients with multidrug-resistant organisms. Klebsiella oxytoca has similar susceptibility patterns as K. pneumoniae and can also produce extended spectrum β-lactamases.

Methods for Typing Escherichia coli strains

Genome sequence of enterohaemorrhagic Escherichia coli O157:H7

Escherichia coli O157:H7 EDL933 Genome

Genome Properties Report

Genes assigned a role category

Organism Gene List

PFGE method

Organism-------------------Escherichia coli

Recommended lysis enzyme--proteinase K

Restriction enzyme---------XbaI,NotI,SfiI

Approximate no. of--15–20,12–15,15–20
restriction Fragments

Fragments size-10–500,10–1000,10–700
range (kb)

General information

E.coli (CDA)

E.coli (FDA)

Identification of Enterobacteriaceae

E.coli 0157

Escherichia coli can be classified by their characteristic virulence properties and different mechanisms that cause disease.

A. Enteroaggregative E. coli (EAEC)
B. Enterohemorrhagic E. coli (EHEC)
C. Enteroinvasive E. coli (EIEC)
D. Enteropathogenic E. coli (EPEC)
E. Enterotoxigenic E. coli (ETEC)

All of the above classes of E. coli cause diarrhea.However, only EHEC produce a verotoxin that has many properties that are similar to Shiga toxin.
EHEC has been associated with hemorrhagic colitis,a severe form of diarrhea, and with HUS. HUS is a disease resulting in acute renal failure, microangiopathic hemolytic anemia, and thrombocytopenia.

Escherichia coli 0157:H7 strains are classically associated with outbreaks of diarrhea after ingestion of undercooked hamburger at fastfood restaurants. Many cases of hemorrhagic colitis and its associated complications can be prevented by thoroughly cooking ground beef.The other organisms listed can cause GI disturbances;
however, E. coli is the classic disturbing pathogen in this case.

Methods for Typing Pseudomonas aeruginosa strains

Complete genome sequence of Pseudomonas aeruginosa PA01

Pseudomonas aeruginosa PA01 Genome Page

Gene assigned a role category

Pseudomonas Genome Data Base

Genome Data base

PFGE

Pseudomonas aeruginosa PFGE protocol (HPA.UK)

ARPAC Pseudomonas aeruginosa Typing Database

PFGE method

Organism-------Pseudomonas aeruginosa

Recommended lysis enzyme--proteinase K

Restriction enzyme----------------SpeI,XbaI

Approximate no. of-----------20–25,40–50
restriction Fragments

Fragments size-------------10–700,10–300
range (kb)

Identification of glucose non-fermenting Gram-negative rods

Pseudomonas


Pseudomonas species is a ubiquitous, aerobic gram-negative bacillus.

Reservoirs in nature include soil, vegetation, and water.

Reservoirs in a hospital include sinks, toilets, mops, respiratory therapy, and dialysis equipment.

It exhibits intrinsic resistance to many antibiotics and disinfectants.
Pseudomonas adheres to host cells by pili and nonpili adhesins. It produces a polysaccharide capsule that allows the organism to adhere to epithelial cells, inhibits phagocytosis,and confers protection against antibiotic activity.

Patients with cystic fibrosis are more likely to be infected with a strain whose colony appears mucoid because of the presence of the capsule.
Pseudomonas produces multiple toxins and enzymes, which contribute to its virulence.Its lipopolysaccharide endotoxin and exotoxin A appear to cause most of the systemic manifestations of Pseudomonas disease. Exotoxin A blocks protein synthesis in host cells, causing direct cytotoxicity. It mediates systemic toxic effects as well. It is similar in function to diphtheria toxin but is structurally and immunologically distinct. Endotoxin contributes to the development of many of the symptoms and signs of sepsis, including fever, leukocytosis, and hypotension.

Antibiotic resistance is another important aspect of its virulence. It is intrinsically resistant to numerous antibiotics and has acquired resistance to others through various means. The polysaccharide capsule prevents the penetration of many antibiotics into Pseudomonas. Penetration of antibiotic into the Pseudomonas cell is usually through pores in the outer membrane. Mutation of these porin proteins appears to be a primary mechanism of its antibiotic resistance. Multidrug efflux pumps and b-lactamase production also contribute to the antibiotic resistance that so frequently complicates the treatment of Pseudomonas infections.

Some P. aeruginosa strains produce a diffusable pigment: pyocyanin, which gives the colonies a blue color; fluorescein, which gives them a yellow color; or pyorubin, which gives them a red-brown color. Pyocyanin also
seems to aid in the virulence of the organism by stimulating an inflammatory response and by producing toxic oxygen radicals.

Clinically significant infections with P. aeruginosa should not be treated with single-drug therapy, because the bacteria can develop resistance when single drugs are employed. The newer quinolones, including ciprofloxacin, are active against Pseudomonas. Quinolones inhibit bacterial DNA synthesis by blocking DNA gyrase. The fluorinated forms of ciprofloxacin and norfloxacin have low toxicity and greater antibacterial activity than the earlier forms. Plasmids code for enzymes that determine the active transport of various antimicrobials across the cell membrane.

Methods for Typing Staphylococcus aureus strains

From " Complete genomes of two clinical Staphylococcus aureus strains: Evidence for the rapid evolution of virulence and drug resistance"

The size of the sequenced S. aureus genomes varies from 2.800 Mbp to 2.903 Mbp, encoding 2565–2721 proteins.
It has been calculated that approx. 75% of the genome is conserved among all S. aureus isolates representing the core genome.

Comparative Genomics of Staphylococcus aureus

Genes of Staphylococcus aureus Genome subsp.aureus Col

Genome Staphylococcus aureus NCTC 8325

Staphylococcus aureus genome sequence

Staphylococcus aureus strain NCTC 8325 annotation list of ORFs

Gene assigned a role category

Harmony

From "Harmony Goals"

Pulsed-Field Gel Electrophoresis

Harmony PFGE protocol


Multilocus Sequence Typing

PFGE method

Organism-------Staphylococcus aureus

Recommended lysis
enzyme:lysozyme,lysostaphin,proteinase K

Restriction enzyme----------------SmaI, CspI

Approximate no. of-----------10–15, 15–20
restriction Fragments

Fragments size-------------10–700, 30–500
range (kb)



Organism-------Staphylococcus(coagulase negative)

Recommended lysis
enzyme:lysozyme,lysostaphin,proteinase K

Restriction enzyme----------------SmaI

Approximate no. of----------- 15–20
restriction Fragments

Fragments size-------------5–400
range (kb)

Identification of Staphylococcus species

Staphylococcus species



Staphylococci belong to the family Micrococcaceae, which includes the genus Micrococcus in addition to Staphylococcus.

Staphylococci grow rapidly on multiple culture media, in a wide range of environments, including up to 10 percent sodium chloride, and in a broad range of temperatures. Staphylococcus aureus is a nonmotile, nonspore forming, facultative anaerobic grampositive coccus that commonly colonizes healthy humans and is a frequent cause of disease.

It is frequently identified as growing in clusters or clumps.
This is a result of the effect of bound coagulase (“clumping factor”), which binds fibrinogen, converts it to insoluble fibrin, and results in aggregation.

Staphylococcus aureus is the only Staphylococcus found in humans which produces coagulase; other staphylococcal species are commonly identified as coagulase-negative staphylocci.

Staphylococcus aureus produces at least five cytolytic toxins, two exfoliative toxins, eight enterotoxins, and toxic shock syndrome toxin. Some of these toxins act as superantigens, which recruit host defense cells that liberate cytokines and, therefore, produce systemic effects.

Heating will kill the S. aureus organisms, but not inactivate the enterotoxins, because they are stable to heating at 100°C (112°F) for 30 minutes and are resistant to breakdown by gastric acids.

Of growing public health concern is the rapid spread of antibiotic resistance within S. aureus isolates.
Some S. aureus isolates have been identified recently with reduced sensitivity to vancomycin. The mechanism of this resistance is unknown.

Genes that confer resistance can be transferred between organisms by plasmid transfer, transduction and cell-to-cell contact.

Typing Methods

Definitions of the Main Terms Describing Genotypic and Epidemiological Relationships Among Isolates

Adapted from Tenover, F.C., Arbeit, R.D., Goering, R.V., Mickelsen, P.A., Murray, B.E., Persing, D.H., and Swaminathan,B.J. Clin. Microbiol., 33(9), 2233–2239, 1995 [3] and Arbeit, R.D.IDCP,5(4), 260–264, 1996 4].

Isolate-Pure culture of bacteria obtained by subculture of a single colony from a primary isolation plate and presumed to be derived from a single organism.

Strain-Isolate or set of isolates that can be differentiated from other isolates of the same genus and species by phenotypic and/or genotypic characteristics. A strain is a relative concept, a descriptive subdivision of a species based on one or more analyses, and therefore should not be used as a synonym for isolate.

Epidemiologically related isolates-Isolates cultured from specimens taken from patients or the environment during a specific time frame or from a particular area as part of an epidemiological investigation that suggests that the isolates may be derived from a common source.

Genetically related isolates (clones)-Isolates that are indistinguishable from each other by a variety of genetic tests or that are so similar that they are presumed to originate from a common precursor.Such isolates are often referred to as having a particular “genotype”, representing a specific “genetic lineage” or to be “clonal.”

Outbreak-Increased incidence of an infectious disease in a specific place during a given period that is above the baseline rate for that place and time frame.

Outbreak strains- Isolates of the same species that are both genetically and epidemiologically related (e.g., by time, place, and common source); by implication, the outbreak represents a set of isolates that are derived from a common parent.

Endemic strains- Isolates that are recovered frequently from infected or colonized patients in a particular place and that are indistinguishable or closely related to each other by typing methods, but for which no direct or epidemiological relationship can be identified.



From "Guidelines for the validation and application of typing methods for use in bacterial epidemiology"

Common Genotyping Methods

-Plasmid Analysis

-Restriction Endonuclease Analysis

-PCR Assays

-Multilocus Enzyme Electrophoresis (MLEE)

-Multilocus Sequence Typing (MLST)

-Pulsed-Field Gel Electrophoresis (PFGE)

-DNA sequencing

-Ribotyping

-PCR Ribotyping

-Restriction Fragment Length Polymorphism (RFLP) studies

-Randomly Amplified Polymorphic DNA (RAPD)

-Amplified Fragment Length Polymorphism (AFLP)

-Repetitive Sequence–Based PCR (rep-PCR)

Pulsed-Field Gel Electrophoresis

Steps:

-Cell lysis and release of intact chromosomal DNA

-Restriction endonuclease digestion of chromosomal DNA

-Separation of the DNA fragments

-Analysis of DNA fragment length polymorphism



PFGE: pulsed-field gel electrophoresis protocol
(M.E.Kaufmann)

From"Pulsed-Field Gel Electrophoresis (PFGE) Technique and its use in Molecular Biology"

PFGE Performance Characteristics

The performance of strain typing technology, including PFGE, is measured by the following criteria:

-Discriminatory power describes the probability that indistinguishable or closely related strains are truly clonal and part of the same chain of transmission. This parameter can be calculated based on Simpson’s index of diversity, using an index greater than 0.95 as acceptable.

-Reproducibility is the ability to obtain the same results upon repeat testing of the same strain.

- Stability is measured by the ability of clonal isolates to consistently express particular markers over time.

-Typeability measures the proportion of isolates within a bacterial species that can be designated a genotype by a molecular typing system.

Restriction Endonuclease Digestion of Chromosomal DNA

SmaI, recognizes the CCC/GGG sequence that cleaves the DNA of most Gram-positive bacteria. SmaI is able to cleave DNA at rarely occurring sites due to the low GC content and AT-rich sequences in Gram-positive bacteria.

XbaI recognizes the T/CTAGA sequence that cleaves that of many Gram-negative bacteria.



From: J Clin Microbiol. 2003 November; 41(11): 4961–4965.
doi: 10.1128/JCM.41.11.4961-4965.2003.

PFGE profiles of 12 isolates of P. mirabilis from mid-stream urine samples, blood samples, and wound swabs from patients in a district general hospital. The isolates in lanes 7 and 8 were from
the urine and blood, respectively, of the same patient. Lanes M, molecular size marker. Relevant sizes of bands are indicated in kilobases.

PFGE is widely used for genotypic characterization of microorganisms. It has been considered the method of choice for the analysis of most bacterial pathogens because of its high discriminatory power and reproducibility. Although PFGE is an important tool for outbreak investigations, it is not indicated for population
analysis of microorganisms. In addition, PFGE is marginally valuable in epidemiological settings of long-term duration encompassing years or decades or at a country to continent level. Its limitations also include substantial measurements of time and resources, and the non-typeability of some strains in some situations. Therefore, a combination of PFGE and PCR-based techniques may be necessary for genotyping a wide range of microorganisms under these circumstances.

PCR-Based Strain Typing Techniques

PCR-based strain typing techniques are designed to generate multiple bands that provide a unique fingerprint for a particular species or strain of microorganism. Unlike diagnostic tests that determine presence or absence of a microorganism (or its nucleic acid) in a specimen,these procedures are used to differentiate epidemiologically unrelated organisms at the species or subspecies level. They must generally produce multiple DNA bands to provide sufficient discrimination power, and these banding patterns must be reproducible run-to-run and among isolates of the same predefined group while clearly distinguishing isolates that epidemiologically or phenotypically fall outside of that group.