PATHOGENS OF ANIMALS AND HUMANS PHOSPHOLIPASE D PRODUCERS AND

THEIR DIAGNOSTIC AND THERAPEUTIC FAILURES

Ljiljana Suvajdžić1 and Zoran Suvajdžić2 1 Faculty of Medicine, Department of

Pharmacy, University of Novi Sad 2Veterinary infirmary, STRIKS, Čerević

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INTRODUCTION

• Phospholipase D is an enzyme that destroys the membrans of mammalian cells and only three bacterial species can produce it.

• Thus, confirmation of this enzyme is a crucial diagnostic parameter (Součkova and Souček 1972; Barksdale et al., 1981).

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• Arcanobacterium haemolyticum,

• Corynebacterium pseudotuberculosis and

• Corynebacterium ulcerans

• From 1982 Arcanobacterium haemolyticum

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ARCANOBACTERIUM HAEMOLYTICUM

• Arcanobacterium haemolyticum predominantly attacks upper respiratory tract of human population.

• but can produce erythrogenic toxin, in which case it

can clinically mimic scarlatina, exanthema toxialergicum and rash fever.

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• The organism rarely causes severe health problems such as sepsis (Ford et al., 1995),

• endocarditis (Worthington et al., 1985),

• mixed wound infections (Barker et al., 1992), neurological complications (Chandrasekar and Molinari 1987)

• and cavitary pneumonia (Waller et al., 1991).

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• Its rare identification is mostly misidentification due to the colonial "mimicry" to another species,

Tuerperella pyogenes, Streptococcus spp. and Listeria spp.

Is it important to know the right causative agent?

Yes, it is, in order to apply rational antibiotic therapy.

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That misidentification leads to diagnostic and therapeutic failures,

increasing the number of hospital days and time spent on sick leave (Clarrindge 1989; Kovatch et al., 1983;

Suvajdzic 2006).

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Pharyngitis

• In our country, a case of seventeen years old teenager was reported (Suvajdzic et al 2006.)

• The patient had mild symptoms of pharyngitis, marked urticarial rash and heavy desquamation of palms and soles that clinicaly mimic scarlatina

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• According to the antibiogram and bacteriological diagnosis of Streptococcus non A non B group, the patient was treated with penicillin; however, ineffectively.

PENICILLIN INEFFECTIVENESS

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Escalation of urticaria: corticosteroids and antihistamines

• Escalation of urticaria and failure of the initial penicillin therapy shifted the diagnosis towards exanthema toxialergicum and thus to the treatment with corticosteroids and antihistamines, yet with no improvement. 10

• Repeated throat swab confirmed the identity of Arcanobacterium haemolyticum.

• Erythromycin 500 mg, twice a day for seven days, resulted in complete eradication of the causative

agent. The patient fully recovered (Suvajdžić et al., 2006).

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• A. haemolyticum rarely causes disease in animals, or at least it is rarely isolated. There are only few cases reported from animal samples.

• The lungs are the most commonly infected organs (Roberts 1969; Suvajdžić 2000; Suvajdžić et al., 2002; Suvajdžić et al., 2012),

• but it was also reported from bull semen (Richardson and Smith 1968)

• and the central nervous system of goat (Younan and Drescher 1996).

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CORYNEBACTERIUM PSEUDOTUBERCULOSIS AND

CORYNEBACTERIUM ULCERANS

• C. pseudotuberculosis is a well-known animal pathogen, with rare reports in humans.

• Epidemiological data for C. ulcerans are inverted.

widespread well-known pathogens

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most frequently sheep.... (Queen et al., 1994; LeaMaster et al., 1987; Brown and Olander 1987; Pepin et al., 1997)

WHO CAN BECOME ILL?

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• and horses (Merchant 1935; Aleman et al., 1982-1983) but

• the other mammals are prone to infection as well.

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• The organism was isolated from goats

(Brown and Olander 1987; Gezon et al., 1991),

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pigs

• (Suvajdžić 2000; Biberstenin and Zee, 1990),

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Cattle

• (Suvajdžić 2000; Yeruham et al., 1996; Songer et al., 1988)

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camels (Songer et al., 1988)

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and humans

• (Jones and Collins 1986).

• Skin wounds are the most common entry portal of infection (Aleman et al., 1982-1983) while the skin and lymphoid tissue is usually its target (Biberstein and Zee, 1990).

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• .

Animal feed, especially if it is not properly stored, could be a source of infection.

The organism can be transmitted by insects (Aleman et al., 1982-1983; Yeruham et al.,

1996), what is important for epidemiological prognosis (Yeruham et al., 1997).

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• depend of pathway of desease spreading. Lymphogenic dissemination is always included (Biberstein and Zee, 1990), but hematogenic and per continuitatem just occasionally (Aleman et al., 1982-1983).

• Pathognomonic are greenish opalescing exudate and

• caseous to dry and crumbly lesions (Biberstein and Zee, 1990).

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Pathogenicity : diphtheria toxin

• In the late seventies and early eighties (Barksdale et al., 1981; Jones and Collins 1986), it became clear that within the Genus Corynebacterium there is a group of related microorganisms able to produce real “diphtheria” toxin.

• If they are lysogenized by beta phage, three bacterial species produce diphtheria toxin

• Corynebacterium diphtheriae

• Corynebacterium pseudotuberculosis

• Corynebacterium ulcerans

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• Cases of skin manifestations (in the form of gangrenous dermatitis) (Olson et al., 1988)

• and necrotizing pneumonia, where C. ulcerans was

• isolated as a monoculture, are described 24

• In abscence of beta phage, the organisms produce only their own toxin, the ovis toxin.

• Its main component is phospholipase D.

• Determination of ovis toxin structure has ended a half-century long speculation about the nature of this toxin (Barksdale et al., 1981).

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• From an epidemiological point of view, it is important to emphasize that C. ulcerans could be transferred to humans by milk and dairy products.

• This microorganism was the only causative agent of food borne outbreaks associated with milk consumption in two of the 27 cases in England and Wales (1983;1984).

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• This indicates the importance of mandatory ruling out the presence of C ulcerans in milk samples in cases of mastitis, as well as in consumable milk and milk products.

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MICROBIOLOGICAL DIAGNOSIS

• .

complicated and expensive

except ......

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TREATMENT AND CONTROL IN HUMANS

• At very begining penicillin was the drug of choice (symptoms withdrawal three days after the introduction of penicillin therapy)

but numerous reports indicated faliures in its per oral and parenteral administration.

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• Banck described 18 patients treated with penicillin V per os 25 mg per kg a day in two daily doses during seven to ten days. Patients had A. haemolyticum in the throat 2 to 4 weeks after therapy (Banck and Nyman, 1986).

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Faliuresof per oral Th

• Based on the high level of penicillin tolerance in 40 isolates, Nyman found that penicillin V is ineffective in the treatment of A. haemolyticum (Nyman et al., 1990).

• Osterlund is of the same opinion, interpreting that with the intracellular survival of microorganisms (Osterlund 1995).

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• Uniform in vitro sensitivity to erythromycin (Carlson et al., 1994) and an excellent effect in clinical practice qualifies erythromycin as antibiotic of choice for treatment of A. haemolyticum infections.

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Erythromycin therapy protocol

• Erythromycin has proven to be effective in oral administration of 250 mg four times a day during ten days and at a dose of 500 mg twice a day during seven days.

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TREATMENT AND CONTROL IN ANIMALS

In sheep and goats penicillin is not efficient. Prevention of disease spreading is limited to the

separation of sick animals, limiting the exposure of infection, sanitary care and hygiene measures.

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Bacterin-toxoid

Bacterin-toxoid combination could be useful in infection limiting.

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Abscesses are treated surgically

Apsces treated surgicaly with local antibiotic administration.

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GENTAMICIN

According to our experience, local administration of gentamicin gives better results than penicillin,

which is consistent with the experience in human medicine.

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REASONS FOR DIAGNOSTIC WANDERING AND HOW TO AVOID

THEM

• What is this all about? Colonies of A. haemolyticum resemble beta hemolytic species of the Genus Streptococcus , Listeria and T. pyogenes colonies.

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• In bacteriological jargon, all of the mentioned genera and species are known as "β small". Thus, a veterinary bacteriologist will "see“ what he expected: T. Pyogenes or Listeria sp..

"β small"

• Medical microbiologist will direct this isolate to the "bacitracin, CAMP test” and, the next day, find it to be Streptococcus non A non B group...

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For the diagnosis of C. ulcerans and C. pseudotuberculosis, there is an even bigger trap: colonies can mimic species of the Genus Staphylococcus. Microbiologist, medical or veterinarian, will forward such colonies to plasma coagulation in a test tube.

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• Since plasma in a tube test will be positive, every microbiologist will believe that he proved "coagulase positive staphylococcus". ...

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• C. pseudotuberculosis could "pass" as species of Genus Staphylococcus, Nocardia-like organism or

diphtheroids.

• Sometimes we declare unrecognized colonies as "Luft bacteria” (air bacteria), which implies

insignificant contamination.

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• For diagnosis of C. pseudotuberculosis clinicians commonly alert microbiologists... because of tipical

“cheesy gland”

but not for C. ulcerans and A. haemolyticum.

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HOW TO AVOID DIAGNOSTIC WANDERING?

BY USING A DOUBLE CAMP TEST! 44

MICROBIOLOGICAL DIAGNOSIS THE INVERSE CAMP

• Zaharova and Kubelka (1960) found that some bacteria produce substances that protect erythrocytes against lysis by staphylococcal toxin.

• They named this phenomenon “the inverse CAMP phenomenon”

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• In the following years and decades, this was confirmed by numerous researchers:

• Souckova and Soucek (1972), • Lamler and Blobel (1988), • Comman (1996), and

• JE Claridge (1989;1995). • Suvajdzic et al. through serial reports during the period 1996-

2014. 46

Rhodococcus CAMP

• Based on synergistic hemolysis with equi factors of Rhodococcus equi Jill E. Claridge developed in 1989. a simple and reliable test that is performed on a single blood agar plate. Suvajdzic et al. through serial reports during the period 1996-2014 confirmed it.

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CONLUSION: DOUBLE CAMP IS SUPREME TOOL

for proving the identity of all three phospholipase D producers 48

COST-BENEFET

• So each β small colony and atypical Staphylococcus- and Nocardia-like colonies, needs to be examined also in a double-CAMP testing that have to become everyday routine.

• At the price of one blood agar plate, we can confirm diagnosis for all three phospholipase D producers, as well as few other species that are rarely found in clinical specimens.

• We should not forget that we can find only what we know to look for.

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Recomendation

• Corynebacterium ulcerans and Corynebacterium pseudotuberculosis have to be mandatory excluded from consume milk and dairy products for the sake of human health.

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