Abstracts

Inhalt

• Zhang JR, Antigenic variation in Lyme disease borreliae by promiscuous recombination of VMP-like sequence cassettes.
• Zhang JR, Norris SJ , Kinetics and in vivo induction of genetic variation of vlsE in Borrelia burgdorferi.
• LymeAlliance.org, LAB TESTS: Reasons Why A Seronegative Test Result Might Occur.
• Bauernfeind et al., In vitro activity and stability against nova beta-lactamases of investigational beta-lactams in comparison with established compounds.
• Wiedemann B, Dietz H, Cefotaxime: Unchanged Antibacterial Activity over Years?
• Klassifikation der beta-Lactamasen nach Bush
• Sanders CC, Das veränderliche Bild der mikrobiellen Resistenz.
• Brorson O et al., Mattman L, Preac Mursic et al., Zystische Formen der Borrelia burgdorferi
• Riedel M et al., Lyme disease presenting as Tourette's syndrome.
• Frieling JTM et al., Differential Induction of Pro- and Anti-Inflammatory Cytokines in Whole Blood by Bacteria: Effects of Antibiotic Treatment.
• Hauser U et al., Validity of interpretation criteria for standardized Western blots (immunoblots) for serodiagnosis of Lyme borreliosis based on sera collected throughout Europe.
• Robertson J; Guy E; Andrews N; Wilske B; Anda P; Granstrom M; Hauser U; Moosmann Y; Sambri V; Schellekens J; Stanek G; Gray J, A European Multicenter Study of Immunoblotting in Serodiagnosis of Lyme Borreliosis.
• Modai J, Diffusion of 3-quaternary ammonium cephem antibiotics into cerebrospinal fluid of patients with bacterial meningitis.
• Wise R, Tissue penetration of the fourth generation parenteral cephalosporins.
• Pachner A et al., 2001, Lyme Borreliosis in Rhesus Macaques: Effects of Corticosteroids on Spirochetal Load and Isotype Switching of Anti-Borrelia burgdorferi Antibody.
• Excerpt from Chapter by Alan Steere in: Principles and Practice of Infectious Diseases, Mandell, Douglas, and Bennett (eds.)

Antigenic variation in Lyme disease borreliae by promiscuous recombination of VMP-like sequence cassettes.

Zhang JR, Hardham JM, Barbour AG, Norris SJ

We have identified and characterized an elaborate genetic system in the Lyme disease spirochete Borrelia burgdorferi that promotes extensive antigenic variation of a surface-exposed lipoprotein, VlsE. A 28 kb linear plasmid of B. burgdorferi B31 (lp28-1) was found to contain a vmp-like sequence (vls) locus that closely resembles the variable major protein (vmp) system for antigenic variation of relapsing fever organisms. Portions of several of the 15 nonexpressed (silent) vls cassette sequences located upstream of vlsE recombined into the central vlsE cassette region during infection of C3H/HeN mice, resulting in antigenic variation of the expressed lipoprotein. This combinatorial variation could potentially produce millions of antigenic variants in the mammalian host.

Kinetics and in vivo induction of genetic variation of vlsE in Borrelia burgdorferi.

Zhang JR, Norris SJ

The Lyme disease agent, Borrelia burgdorferi, is able to persistently infect humans and animals for months or years in the presence of an active immune response. It is not known how the organisms survive immune attack in the mammalian host.

BACKGROUND:

vlsE, a gene localized near one end of linear plasmid lp28-1 and encoding a surface-exposed lipoprotein in B. burgdorferi B31, was shown recently to undergo extensive genetic and antigenic variation within 28 days of initial infection in C3H/HeN mice.

METHODS:

In this study, we examined the kinetics of vlsE sequence variation in C3H/HeN mice at

• 4 days,
• 7 days,
• 14 days,
• 21 days,
• 28 days,
• 7 months and
• 12 months

RESULTS:

1. Sequence changes were detected by PCR amplification and sequence analysis as early as 4 days postinfection and accumulated progressively in both C3H/HeN and CB-17 severe combined immunodeficient (SCID) mice throughout the course of infection.
2. The sequence changes were consistent with sequential recombination of segments from multiple silent vls cassette sites into the vlsE expression site.
3. No vlsE sequence changes were detected in organisms cultured in vitro for up to 84 days.

1. These results indicate that vlsE recombination is induced by a factor(s) present in the mammalian host, independent of adaptive immune responses.
2. The possible inducing conditions appear to be present in various tissue sites because isolates from multiple tissues showed similar degrees of sequence variation.

The rate of accumulation of predicted amino acid changes was higher in the immunologically intact C3H/HeN mice than in SCID mice, a finding consistent with immune selection of VlsE variants.

http://www.lymealliance.org/Medical/MedCategory4/Med24/med24.html

LAB TESTS: Reasons Why A Seronegative Test Result Might Occur

The Lyme Alliance

1. Recent infection before immune response
2. Antibodies are in immune complexes
3. Spirochete encapsulated by host tissue (i.e. lymphocytic celI walls)
4. Spirochete are deep in host tissue
5. Blebs in body fluid, no whole organisms needed for PCR
6. No spirochetes in body fluid on day of test
7. Genetic heterogeneity (300 strains in U.S.)
8. Antigenic variability
9. Surface antigens change with temperature
10. Utilization of host protease instead of microbial protease
11. Spirochete in dormancy phase
12. Recent antibiotic treatment
13. Recent anti-inflammatory treatment
14. Concomitant infection with babesia may cause immunosuppression
15. Other causes of immunosuppression
16. Lab with poor technical capability for Lyme disease
17. Lab tests labeled "for investigational use only"
18. Center for Disease Control criteria is epidemiological, not a diagnostic criteria

In vitro activity and stability against nova beta-lactamases of investigational beta-lactams in comparison with established compounds

Bauernfeind et al.

Werte sind Minimale Hemmkonzentrationen (MHK) in mg/l

Typ der beta-Lactamase	Cefepim	Cefpirom	Cefotaxim	Cefoxitin	Piperacillin	Pip./ Tazobactam
TEM-1	0.15	0.06	0.06	4	>64	16
TEM-2	0.03	0.13	0.06	4	16	8
TEM-3	2	4	32	16	>64	8
TEM-4	4	4	32	8	kein Wert	kein Wert
TEM-5	0.5	1	2	16	32	8
TEM-6	4	2	2	4	>64	16
TEM-7	0.5	1	0.25	8	>64	8
SHV-2	8	16	32	8	>64	32
SHV-3	2	8	32	16	kein Wert	kein Wert
SHV-4	2	16	32	4	>64	64
SHV-5	4	8	32	4	>64	32
CMY-1	0.25	2	>64	>64	>64	32
CTX-M-1	16	16	>64	8	>64	16

Diagn Microbiol Infect Dis 1995; 22:5-12
Elsevier Science Inc, 655 Av of the Americas, New York, NY 10010, USA

Cefotaxime: Unchanged Antibacterial Activity over Years?

Wiedemann B, Dietz H

Tab. 1: Auftreten von Extended-Spectrum beta-Lactamasen

Jahr	Type	Klasse	Repräsentative Spezies	Genetischer Ursprung
1978	AmpC	C	E. cloacae, Citrobacter freundii, Pseudomonas aeruginosa	Chromosome
1983	SHV-2, TEM-3	A	Klebsiella pneumoniae, E. coli, C. freundii	Plasmid
1986	K1	A	Klebsiella	Chromosome
1990	AmpC	C	K. pneumoniae, E. coli	Plasmid
1993	Metalloenzyme	B	P. aeruginosa	Plasmid
1993	OXA-11	D	P. aeruginosa	Plasmid

Anmerkung: Klassen A - D nach einer Einteilung, die auf der Aminosäuren-Sequenz beruht.

Tab. 2: Klassifikation der beta-Lactamasen nach Bush, 1989

Bush-Klasse	Enzym-Typ	Representative Enzyme
1	Cephalosporinase	Chromosomal Gram-negativ
2a	Penicillinase	Staphylococcus aureus, Pseudomonas aeruginosa
2b	Breitband-Spektrum	TEM-1, -2, SHV-1
2b'	Extended Spectrum	TEM-3, -10, SHV-2, -5
2c	Carbenicillinase	PSE, CARB
2d	Cloxacillinase	P. aeruginosa
2e	Cephalosporinase	Chromosomal Proteus vulgaris
3	Metalloenzyme	Chromosomal
4	Penicillinase	Chromosomal P. vulgaris

Continuing Education 1996; E4-X005: 1-8
Bristol-Myers Squibb Company, Route 206 & Providence Line Princeton, NJ 08543, USA, Tel.: (001) 609 - 252-5141

Das veränderliche Bild der mikrobiellen Resistenz

Sanders, Christie C

...

Die beta-Lactamasen von gram-negativen Bakterien sind vor kurzem in Übersichtsartikeln beschrieben worden:

• Sanders CC. beta-lactamases of gram-negative bacteria: new challenges for new drugs. Clin. Infect. Dis. 1992:14:1089-1099.
• Sanders CC, Sanders WE Jr. beta-lactam resistance in gram-negative bacteria: global trends and clinical impact. Clin. Infect. Dis. 1992:15:824-839.
• Bush K, Jacoby GA, Medeiros AA. A functional classification scheme for beta-lactamases and its correlation with molecular structure. Minireview, Antimicrob. Agents Chemother. 1995; 39:1211-1233.

Obwohl sie ziemlich divers sind, schließen die am häufigsten auftretenden beta-Lactamasen die Enzyme der Bush-Gruppen 1 und 2 ein (siehe Bush's genanntes Minreview).

• Bush-Gruppe-1-Enzyme sind die normalerweise chromosomalen ampC-Enzyme, die nach Induktion vom Bakterium dargestellt werden
• in Spezies von Enterobacter,
• in Spezies von Providencia und
• in Spezies von Serratia, und
• in Citrobacter freundii,
• Morganella morganii und
• Pseudomonas aeroginosa.
Diese Enzyme sind intrinsisch resistent gegen die beta-Lactamasehemmer wie Clavulansäure und sind vorwiegend Cephalosporinasen (beta-Lactamasen, die sich gegen Cephalosporine richten), obwohl sie jede der Hauptklassen-beta-Lactamase- Antibiotika hydrolysieren.
• Bush-Gruppe-2-Enzyme sind die am häufigsten angetroffenen Plasmid-kodierten beta-Lactamasen, TEM-1, TEM-2, SHV-1 und die mutierten Derivate dieser Enzyme, die extended-spectrum beta-lactamases (ESBL's), die sich aus ihnen entwickelt haben. Die Bush-Gruppe-2-Enzyme sind intrinsisch empfindlich gegen beta-Lactamase-Hemmer, obwohl mutierte Formen beschrieben werden, die unempfindlich gegen die Hemmer sind.
• Bush-Gruppe-3-Enzyme schliessen die weniger häufig anzutreffenden metallo-beta-Lactamasen, die für die Resistenz gegen Carbapeneme verantwortlich sind (siehe Bush's genanntes Minreview).

Es gibt drei Haupt-Mechanismen, durch die Organismen antimikrobiellen Mitteln widerstehen können (Holmerg SD, Solomon SL, Blake PA. Health and economic impacts of antimicrobial resistance. Rev. Infect. Dis 1987:9:1065-1078.)

1. Verhinderung der Ansammlung toxischer Konzentrationen der Droge in der Zelle
• Veränderungen an den Proteinen der äußeren Membran (nur gram-negative Bakterien haben eine solche). Beispiel: Imipenem-Resistenz bei Pseudomonas aeruginosa.
• Verminderter aktiver Transport der Droge in die Zelle hinein. Beispiel: Aminoglycosid-Resistenz bei gram-positiven und -negativen Bakterien.
• Aktiver Transport der Droge aus der Zelle heraus. Beispiel: Tetracyclin oder Quinilon-Resistenz bei gram-positiven oder -negativen Bakterien.
2. Veränderung des Zielorts der Droge
• Erwerb eines neuen, weniger empfindlichen Zielorts. Beispiel: Erwerb des PbP 2a (Penicillin bindendes Protein 2a) in Methicillin-resistenten Staphylococcen.
• Mutation des Zielorts hin zu einer weniger empfänglichen Form. Beispiel: veränderte DNA-Gyrase in Quinolon-resistenten gram-positiven und -negativen Bakterien.
• Erwerb von vielfachen neuen Genen, die den Zielort verändern. Beispiel: Vancomycin-Resistenz bei Enterococcen.
3. Produktion von inaktivierenden Enzymen
• Chloramphenicol-Azetyltransferase,
• Aminoglycosid-inaktivierende Enzyme,
• beta-Lactamasen.

....

Resistenz über die Undurchläßigkeit der äußeren Membrane ist ein wirksamer Resistenz-Mechanismus gegen beta-Lactam-Antibiotika bei gram-negativen Bakterien, insbeondere in der Kombination mit beta-Lactamasen.
 
 

Sepsis und Antibiotika-induzierte Freisetzung von Endotoxinen: Konsequenzen für die Therapie?

Heinemann M, Trautmann M,

Abt. für Medizinische Mikrobiologie und Hygiene, Universitätsklinikum Ulm, Steinhövelstr. 9, 89075 Ulm
matthias.trautmann@medizin.uni-ulm.de

....

Die essentiellen Penicillin bindenden Proteine (PbP) werden ihrem Molekulargewicht entsprechend als PbP-1, -2 und -3 bezeichnet. Die Blockade nur eines der 3 essentiellen PbP führt nicht notwendigerweise zum Absterben der Bakterienzelle (siehe Abb. 1 in Heinemann M, Trautmann M, 1999)

1. Die Antibiotika-Interaktion mit PbP-1 in einer Konzentration oberhalb der Minimalen Hemmkonzentration (MHK) führt zu einem relativ schnellen Absterben der Bakterien (Prins et al. 1994). Cefsulodin und Cephaloridin sind paradigmatisch für Antibiotika, die primär an PBP-1 binden und rasch bakterizid wirken.
2. Beta-Lactam-Antibiotika, die eine hohe PBP-2-Affinität aufweisen, führen zur Bildung von rundlichen Zellen, den so genannten Sphäroplasten ohne einen allzu ausgedehnten Zellwandzerfall. Dadurch bleibt die Endotoxin-Freisetzung begrenzt (Prins et al. 1994, Trautmann et al. 1998, Pucci et al. 1991). Carbapeneme wie Imipenem und Meropenem sind typische Vertreter von PBP-2-Inhibitoren [50]. Zu den PBP-2- spezifischen Antibiotika, welche die Bil- dung von Sphäroplasten induzieren, gehören außerdem Mecillinam, Clavulansäure und Cefepim, ein Cephalosporin der vierten Generation (Prins et al. 1994, Pucci et al. 1991).
3. Antibiotika mit selektiver PBP-3-Aktivität stören zunächst nur die bakterielle Septierung und führen daher zur Bildung von langen, filamentösen Zellen. Beispiele sind: Aztreonam, Piperacillin, Mezlocillin und bei niedrigeren Antibiotika- Konzentrationen auch Cefuroxim und die Cephalosporine der dritten Generation, Ceftazidim und Cefotaxim Mehrere Arbeitsgruppen konnten zeigen, dass die Lyse filamentõser, bakterieller Zellen zu einer schnellen und deutlichen Zunahme der Endotoxin-Freisetzung führt, vermutlich aufgrund der großen Biomasse dieser langen Zellen (Hurley 1992, Trautmann 1998, Trautmann 1998, Hurley 1993).

Hurley JC. Antibiotic-induced release of endo- toxin: a reappraisal. Clin Infect Dis 1992;15: 840-54.
Hurley JC. Reappraisal of the role of endotoxin in the sepsis syndrome. Lancet 1993;341:1133- 5.
Prins JM, van Deventer SJ, Kuijper EJ, Speel- man P Clinical relevance of antibiotic-induced endotoxin release. Antimicrob Agents Chemo- ther 1994;38:1211-8.
Pucci MJ, Boice-Sowek J, Kessler RE, Doug- herty TJ. Comparison of cefepime, cefpirome, and cefaclidine binding affinities for penicillin- binding proteins in Escherichia coli K-12 and Pseudomonas aeruginosa SC8329. Antimicrob Agents Chemother 1991;35:2312-7.
Trautmann M, Zick R, Rukavina T, Cross AS, et al. Antibiotic-induced release of endotoxin: in- vitro comparison of meropenem and other anti- biotics. J Antimicrob Chemother 1998;41:163- 9.
Trautmann M, Heinemann M, Zick R, Moricke A, et al. Antibacterial activity of meropenem against Pseudomonas aeruginosa, including an- tibiotic-induced morphological changes and endotoxin-liberating effects. Eur J Clin Micro- biol Infect Dis 1998;17:754-60.

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Zystische Formen der Borrelia burgdorferi

Borrelia burgdorferi Cystic Forms

Mattman 1993, Preac Mursic et al 1996, Brorson & Brorson 1998, Burgdorfer 1999, N.M. Ovcinnikov and V.V. Delectorskij 1968/71, Atlas R, Joanne Rubel (ed.)

Aussehen - Appearance

Fig. 1: Schema einer Spirochete mit Zyste:

1. Querschnitt und
2. Draufsicht (unten links)

(click here for English text).

Das Verhältnis von Zysten- und Spirocheten-Volumen variiert in weitesten Grenzen,

• von kleinen knospenähnlichen Zysten an im wesentlichen intakten Spirocheten

Preac-Mursic et al. 1996, veröffentlichten in der Zeitschrift Infection eine entsprechende Scanning-Electron-Mikroskop-Aufnahme, die W. Burgdorfer als Fig. 14 in The Complexity of Vector-borne Spirochetes (Borrelia spp) auf der 12th International Conference on Lyme Disease and Other Spirochetal and Tick-Borne Disorders, New York City, April 9-10, 1999, vorführte: Borrelia burgdorferi (Bb) wurde in Modified-Kelly-Pettenkofer-Kulturmedium (MKP-Medium) bei 33 C angezüchtet. 48 Stunden nach Zugeben von Penicillin wurde die Aufnahme gemacht: zwei kugelförmige Körper hängen mit einer schwachen Verbindung an der Spirale eines Borrelia-Organismus.
• bis hin zu großen Gebilden mit überhaupt fehlenden oder winzigen Spirocheten-Ansätzen (Brorson, Brorson 1998).

• from small bud-like structures attached to the spirochete

e.g. the spherical bodies cultured by incubation in Modified-Kelly-Pettenkofer-culture medium (MKP-medium) at 33 C, induced by 48 hours exposure to penicillin, scanning electron micrograph by Preac-Mursic et al. 1996 also shown as Figure 14 in: W. Burgdorfer, The Complexity of Vector-borne Spirochetes (Borrelia spp), keynote address, 12th International Conference on Lyme Disease and Other Spirochetal and Tick-Borne Disorders, New York City, April 9-10, 1999.
• to large globs, from which no or tiny spirochetes protrude.)

(Small cysts that have detached from the spirochete are called "blebs").

Die im Vergleich zu Gram-positiven Bakterien sehr dünne Zellwand setzt kleinen Molekülen wie den Antibiotika keinen Durchlaßwiderstand entgegen, während hingegen die äußere und die Zytoplasma-Membran die Durchläßigkeit sehr aktiv bestimmen.

• Für beta-Lactam-Antibiotika ist die äußere Membran eine Barriere.
• Beta-Lactam-Antibiotika binden sich an die Penicillin-bindenden-Proteine (PBP) und die Beta-Lactamasen (beta).
• Die Zielpositionen aller anderen Antibiotika liegen innerhalb der Zytoplasma-Membran. Bakterien können eine Resistenz gegen diese Drogen dadurch entwickeln, daß sie eine Ansammlung der Droge im Inneren der Zytoplasma-Membran verhindern.

Eigenschaften - Properties of cystic forms

1. Preac Mursic et al 1996
• Persistors [i.e. B. burgdorferi found after long antibiotic treatments of the patients] isolated from a great number of patients (60- 80%) after treatment with antibiotics had an atypical form. The morphological alterations in culture with penicillin G developed gradually and increased with duration of incubation.
• Pleomorphism,
• the presence of elongated forms and spherical structures,
• the inability of cells to replicate,
• the long period of adaptation to growth in MKP-medium and
• the mycoplasma-like colonies after growth in solid medium (PMR agar)
suggest that B. burgdorferi produce spheroplast-L-form variants.
• With regard to the polyphase course of Lyme borreliosis, these forms without cell walls can be a possible reason why
• Borrelia survive in the organism for a long time and
• the cell-wall-dependent antibody titers disappear only to emerge again after reversion.
2. Brorson, Brorson 1998, 1999, 2009
• Normal, mobile spirochetes were inoculated into spinal fluid. The spirochetes converted to cysts (spheroplast L-forms) after 1-24 h.
• When these cystic forms were transferred to a rich BSK-H medium, the cysts converted back to normal, mobile spirochetes after incubation for 9 to 17 days, the older the cysts the more time they needed for reversion. If growth medium is insufficient, no reversion was observed.
• Young cysts were observed to be relatively empty except for some spirochete-like structures, whereas in old cysts these structures will be altered towards core structures.
• When cysts are opended forcibly with NaOH, spirochete-like structures will escape from them.
• When neuroborreliosis is suspected, it is necessary to realize that B. burgdorferi can be present in a cystic form, and these cysts have to be recognized by microscopy. This study may also explain why cultivation of spinal fluid often is negative with respect to B. burgdorferi.
• "There is evidence (in vitro) that metronidazol (Flagyl) will kill the cystic form. This fits with the now well known clinical observations that Flagyl can be remarkably effective for many chronic Lyme patients. However, this medication apparently has no effect on intact spirochetes.

Therefore, the trend now is to treat the chronically infected patient who has resistant disease by combining Flagyl with 1 or 2 other antibiotics to target all forms of Bb. Because there is laboratory evidence that tetracyclines may inhibit the effect of Flagyl, this class of medication should not be used in these 2- and 3-drug regimens" (Burrascano, 1999).

• "We report in vitro inhibition and destruction of B. burgdorferi (helices, RBs = "cysts") by the antibiotic Tigecycline (TG; Wyeth), a glycylcycline protein-synthesis inhibitor (of both 30S and 70S ribosome subunits). Studies of the pleiomorphic life history stages in response to TG of both B. burgdorferi and Treponema pallidum in vivo and in vitro are strongly encouraged." Source: Oystein Brorson, Sverre-Henning Brorson, John Scythes, James MacAllister, Andrew Wiere, and Lynn Margulis (alternative email: lynn@sagantechnology.com), "Destruction of spirochete Borrelia burgdorferi round-body propagules (RBs) by the antibiotic Tigecycline", Proc Natl Acad Sci U S A. 2009 Nov 3;106(44):18656-61 (PubMed). more ...

3. Alban PS, Johnson PW, Nelson DR
• B. burgdorferi cells cannot synthesize  fatty acids de novo and serum is thought to provide a source of fatty acids and lipids.
• When B. burgdorferi cells were  serum-starved (in defined RPMI medium), -90% of the cells formed spherical cysts within 48 h.
• Cyst formation was  inhibited by tetracycline.
• Cyst opening and recovery of vegetative cells (cells that are able to divide) was rapidly induced by the addition of either BSKII medium or rabbit serum. 2.9% to 52.5% of cysts converted into viable cells.
• Viability was inversely  proportional to cyst age.
• Data suggest that cells of B. burgdorferi, although possessing a small genome and extremely limited biosynthetic  capabilities, rapidly respond to conditions of serum starvation by inducing changes in protein synthesis and cell  morphology.
4. Ovcinnikov NM, Delectorskij VV 1968, 1971
• Formation of protective cysts is widespread in nature.
• Can the host harbour cysts of pathogenic nature?
5. Gruber 1999
• Theoretically, cystic forms may act as niches protecting Bb from immediate destruction by antibiotics or immune system.
• They release spirochetal forms of Bb (by reversion), and the thus newly developed outer membrane and cell wall components -being TI-1 antigens- can
1. either drive self-organized feedback oscillations of the immune system or
2. lead to cycles of the elimination and inflammation imprinted on the immune system by fluctuations of the endocrine system.

Literatur - Literature

• Cheryl's Lyme Info Web Site, Lyme Files
  1. Cystic Form File:
     These abstracts document the ability of Borrelia burgdorferi (Bb) and related bacteria to"'shape-shift" and change forms in response to surrounding conditions. These cystic forms shed light on the mechanisms Bb uses to survive antibiotics. The fact that Bb does this gives a scientific explanation for what many patients experience...the improvement of their medical condition on antibiotics, worsening when off. 17 pages.
  2. Pictures of Alternate Forms of Spirochetes:
     This file contains photographs of alternate forms of spirochetes -- cystic, granular, bleb forms, etc. Quotations from peer-reviewed studies are included to aid in understanding how the alternate forms enable spirochetes to overcome adverse environmental conditions. The ability of Bb (the Lyme disease bacterium) to transform itself to and from non-spirochetal forms provides a cogent explanation for relapses after antibiotic therapy, and for the occurrence of seronegative Lyme disease. 30 pages. Note: This file is fairly large (4 MB)Ê because it contains a large number of photographs, so be aware that it may take a while to download.
  3. Alt Forms Bibliography:
     This file is a complete bibliography of over 250 studies (!) with information/observations on alternative forms of spirochetes. 48 pages
• Two very extensive literature searches on cystic forms of spirochetes: I, II.
• Cystic Forms: Search Marie Kroun's LymeRICK server

Conversion of Borrelia garinii cystic forms to motile spirochetes in vivo.

Gruntar I, Malovrh T, Murgia R, Cinco M

ABSTRACT  - Cystic forms (also called spheroplasts or starvation forms) andtheir ability to reconvert into normal motile spirochetes have already been demonstrated in the Borrelia burgdorferi sensu lato complex. The aim of this study was to determine whether motile B. garinii coulddevelop from cystic forms, not only in vitro but also in vivo, in cyst-inoculated mice. The cysts prepared in distilled water were able to reconvert into normal motile spirochetes at any time during in vitro experiments, lasting one month, even after freeze-thawing of the cysts. Motile spirochetes were successfully isolated from 2 out of 15 mice inoculated intraperitoneally with cystic forms, showing the infectivity of the cysts. The demonstrated capacity of the cysts to reconvert into motile spirochetes in vivo and their surprising resistance to adverse environmental conditions should lead to further studies on the role and function of these forms in Lyme disease.

Further Study of Ultrathin Section of Treponema Pallidum under the Electron Microscope

N.M. Ovcinnikov and V.V. Delectorskij

That sharply-marked structural elements of the treponeme and its complex and characteristic structure indicate that cysts are not a product of degeneration. In addition, in cultures where there are many cysts, they are very mobile, which is another argument against degeneration. [as an interpretation of cysts]

... A treponeme may undergo transverse division in several places along its length, not merely in one place.

...In actual fact, under unfavourable conditions of existence, treponemes form real cysts as a method of persistent survival and multiplication, as occurs not infrequently among protozoa.

..An important proof of the viability of cysts is their motility, as seen under dark-field and phase-contrast microscopy. When transfers are made from cultures containing cysts and almost no ordinary spiral forms, growth of ordinary spiral forms occurs.

...This phenomenon [the formation of protective cysts] is widespread in nature.
 

• The huge majority of cysts in protozoa, according to Dogel (1951), are a means of protecting their content against unfavourable conditions, but
• some of them are designed rather to ensure a long period of rest.
• Later, depending on conditions and when the harmful exposure is past, protective cysts may become multiplcation cysts.
• They are not of a purely protective character but also serve for multiplcation...
• The main factor regulating cyst formation is the exhaustion of food in the environment.

Current Concepts of the Morphology and Biology of Treponema pallidum based on Electron Microscopy

N.M. Ovcinnikov and V.V. Delectorskij

Of extreme importance is the ability of treponemes to form cysts under unfavourable conditions... Under stressful conditions, the treponeme 'packs' itself into a compact roll (Fig. 8) and becomes covered with a transparent mucoid capsule, which resists the penetration of drugs and antibodies. The organisms may persist in this form for a prolonged period without any reaction from the host. The encysted treponemes and the host coexist more or less peacefully, but under propitious circumstances the cysts may be transformed again into the usual spiral, which damages the cells of the host and elecits a response.

...Moreover, a cyst may contain laminar bodies of variable dimensions; we believe that these are stores of nutrients.

...Can the host harbour cysts of pathogenic treponemes? This is a problem of paramount importance. Even those authors who admit the possibility of encystment of T. pallidum do not consider it likely that cysts may exist in host tissues. By means of electron microscopy we have succeeded in demonstrating the presence of cysts in a rabbit chancre, and we believe that this observation is of considerable significance.

The material was taken from an 8-month-old chancre. When examining the cysts, we could distincly see multi-layered membranes and treponemes cut in various places. Obviously such a membrane serves as a strong barrier to drugs.

...In a rabbit chancre T. pallidum may exist both inside and outside cells.

...The L-forms of T. pallidum do not differ significantly in appearance from those of other organisms, for example gonococcus, Proteus vulgaris, etc. When L-forms are transferred to the usual media they soon reverse to the original forms... Some of them are seen to divide. Thus it is evident that T. pallidum may produce L-forms under conditions of stress. In our experiments the organisms were stressed with penicillin, various drugs, antisera, etc., and the addition of these agents led to the appearance of L-forms. We have not yet isolated L-forms directly from animals affected with syphilis, but undoubtedly they can be present in rabbit tissues.

The significance of these data can hardly be overestimated if we recall that conversion to L-forms alters

• the clinical course of a disease,
• the virulence and pathogenicity of the organisms,
• their antigenic activity,
• drug resistance,

Lyme disease presenting as Tourette's syndrome

Michael Riedel, Andreas Straube, Markus J Schwartz, Betina Wilske, Norbert Müller

A boy at the age of 4 years developed a simple motor tic (blinking) that resoved within a year without treatment. At the age of 9 years, he developed

• multiple orafacial tics including shaking of the head, and several weeks later a vocal tic occurred.
• The tics became exacerbated under stress, as typically seen in Tourette's syndrome.
• Social disabilities such as loss of impulse control, social withdrawl, worsened performance at school followed.

• Serum IgM antibody titres against Borrelia burgdorferi measured by ELISA were not increased;
• although IgG antibody titres (ELISA) were increased at 58 U/mL (normal \< U/ml) and 100 U/mL at another examination 2 weeks later.
• Immunoflourescence absorption test (IFT) was also increased (1:28 [normal \<1:16]).
• IgG immunoblot (4) was positive.

Examination of cerebral fluid

• showed a slight lymphocytic pleocytosis (16 cells per uL) (5) which suggested an inflammatory reaction.
• The CSF: serum IgG ratio for IgG antibodies was 2.0, indicating intraethecal production of B. burgdorferi specific IgG antibodies, as occurs in neuroborreliosis. (4)

• The tics improved after the sixth dose, and
• after the tenth dose the tics resolved completely.
• His social skills returned to normal.
• Follow-up examinations showed no reccurence of tics or other neurological or psychiatric disorder.
• Serum IgG antibody titre tests and IFT tests against B burgdorferi were 11 U/mlL and 1:32 after 1 year.

Persistence of the tics and increasing severity of the social disabilities over several months suggest that the first signs of a Tourette-like syndrome 11 months previously were an expression of an early Lyme infection.

Infection with B burgdorferi should be considered in cases of Tourette's syndrome in endemic areas.

References

1. Shapiro ED, Selzter EG, Lyme disease in children. Semin Neurol 1997;17:39-44.


2. Kaplan RF, Jones-Woodward L,. Lyme encephalopathy; a neuropsychological perspective. Semin Neurol 1997;17:31-37


3. Fallon BA, Nields JA, Parsons B, Liebowitz MR, Klein DF, Psychiatric manifestations of Lyme Borreliosis. J Clin Psychiatry 1997;54:263-68


4. Wilske B, Fingerle V, Herzer P, et al. Recombinant immunoblot in the serodiagnosis of Lyme borreliosis. Med Mikrobiol Immunol 1993; 182:255-70.


5. Pfister HW, Wilske B, Weber K., Lyme borreliosis: basic science and clinical aspects. Lancet 1994; 363:1031-16

Differential Induction of Pro- and Anti-Inflammatory Cytokines in Whole Blood by Bacteria: Effects of Antibiotic Treatment

J. T. M. Frieling, J. A. Mulder, T. Hendrics, J. H. A. J. Curfs, C. J. van der Linden, R. W. Sauerwein

....

LPS wird von lebenden und wachsenden Bakterien abgegeben, aber ein Abtöten der Bakterien durch antimikrobielle Medikamente kann zusätzlich LPS freisetzen. Dies kann zu einem anfänglich schädigenden Einfluß der antibiotischen Behandlung auf den Zustand des Patienten führen. Solche Phänomene wurden bei Typhus (1) und vermuteter Ansteckung durch gram-negative Bakterien beschrieben (2) und werden unterstützt durch Studien, die erhöhte Konzentrationen zeigen nach Antibiotika-Behandlung von

• klinischen (3) oder experimentellen (4 - 7) Infektionen und
• in in-vitro Modellen (8 - 15).

Die Menge von abgegebenem LPS ist durch den Typ und die Konzentration des Antibiotikums bestimmt, ebenso wie durch die Art und Empfindlichkeit der Bakterien. Plasma-LPS-Konzentrationen sind gewöhnlich nicht mit den klinischen Symptomen korreliert (16). Es ist die Erzeugung von Zytokinen durch die Zellwandkomponenten wie LPS, welche die biologischen Antworten während der bakteriellen Infektionen vermitteln. Es wurde wiederholt nachgewiesen, daß Zytokine-Konzentrationen und -Typen mit klinischem Ergebnis korrelieren (17 - 19).

....

LPS ist nur eine der Zellwandkomponenten, die eine entzündliche Antwort hervorrufen können (20). Gram-positive Mikroorganismen, die keine LPS in ihrer Zellwand haben, erzeugen die Freisetzung von Zytokinen durch Peptidoglykane und Teichionsäure (21, 22).

zur mildernden Wirkung von Tetracyclinen siehe

• Whiteman M, Halliwell B, Prevention of peroxynitrite-dependent tyrosine nitration and inactivation of alpha1-antiproteinase by antibiotics., Free Radic Res 1997 Jan;26(1):49-56
• Milano S, Arcoleo F, D'Agostino P, Cillari E, Intraperitoneal injection of tetracyclines protects mice from lethal endotoxemia downregulating inducible nitric oxide synthase in various organs and cytokine and nitrate secretion in blood, Antimicrob Agents Chemother 1997 Jan;41(1):117-21. (vollständiger Artikel in pdf-Format).
• Shapira L, Soskolne WA, Houri Y, Barak V, Halabi A, Stabholz A., Protection against endotoxic shock and lipopolysaccharide-induced local inflammation by tetracycline: correlation with inhibition of cytokine secretion. (vollständiger Artikel in pdf-Format).

1. Buxton Hopkin, D. A.; 1977. Too rapid destruction of gram-negative organisms. Lancet i:603-604. 
2. Shenep, J. L., P. M. Flynn, F. F. Barrett, G. L. Stidham, and D. F. Westenkirchner; 1988. Serial quantitation of endotoxemia and bacteremia during therapy for gram-negative bacterial sepsis. J. Infect. Dis. 157:565-568.
3. Hurley, J. C., W. J. Louis, F. A. Tosolini, and J. B. Carlin; 1991. Antibiotic-induced release of endotoxin in chronically bacteriuric patients. Antimicrob. Agents Chemother. 35:3288-3294.
4. Friedland, I. R., H. Jafari, S. Ehrett, S. Rinderknecht, M. Paris, M. Coulthard, H. Saxen, K. Olsen, and G. H. McCracken; 1993. Comparison of endotoxin release by different antimicrobial agents and the effect on inflammation in experimental Escherichia coli meningitis. J. Infect. Dis. 168:657-662. 
5. Mertsola, J., O. Ramilo, M. M. Mustafa, X. Saez-LLorens, E. J. Hansen, and G. H. McCracken; 1989. Release of endotoxin after antibiotic treatment of gram-negative bacterial meningitis. Pediatr. Infect. Dis. J. 8:904-906. 
6. Mohler, J., B. Fantin, J. L. Mainardi, and C. Carbon; 1994. Influence of antimicrobial therapy on kinetics of tumor necrosis factor levels in experimental endocarditis caused by Klebsiella pneumoniae. Antimicrob. Agents Chemother. 38:1017-1022. 
7. Shenep, J. L., R. P. Barton, and K. A. Mogan; 1985. Role of antibiotic class in the rate of liberation of endotoxin during therapy for experimental gram-negative bacterial sepsis. J. Infect. Dis. 151:1012-1018. 
8. Bingen, E., V. Goury, H. Bennani, N. Lambert-Zechovsky, Y. Aujard, J. C. Darbord; 1992. Bactericidal activity of beta-lactams and amikacin against Haemophilus influenzae: effect on endotoxin release. J. Antimicrob. Chemother. 30:165-172. 
9. Cohen, J., and J. S. McConnell; 1985. Antibiotic-induced endotoxin release. Lancet i:1089-1090. (Letter.) 
10. Cohen, J., and J. S. McConnell; 1986. Release of endotoxin from bacteria exposed to ciprofloxacin and its prevention with polymixin B. Eur. J. Clin. Microbiol. 5:13-17. 
11. Crosby, H. A., J. F. Bion, C. W. Penn, and T. S. J. Elliott; 1994. Antibiotic-induced release of endotoxin from bacteria in vitro. J. Med. Microbiol. 40:23-30.
12. Eng, R. H. K., S. M. Smith, P. Fan-Havard, and T. Ogbara; 1993. Effect of antibiotics on endotoxin release from gram-negative bacteria. Diagn. Microbiol. Infect. Dis. 16:185-189. 
13. Evans, M. E., and M. Pollack; 1993. Effect of antibiotic class and concentration on the release of lipopolysaccharide from Escherichia coli. J. Infect. Dis. 167:1336-1343. 
14. McConnell, J. S., and J. Cohen; 1986. Release of endotoxin from Escherichia coli by quinolones. J. Antimicrob. Chemother. 18:765-766. (Letter.) 
15. Van den Berg, C., A. J. De Neeling, C. S. Schot, W. M. N. Hustinx, J. Wemer, and D. J. de Wildt; 1992. Delayed antibiotic-induced lysis of Escherichia coli in vitro is correlated with enhancement of LPS release. Scand. J. Infect. Dis. 24:619-627. 
16. Roumen, R. M. H., J. T. M. Frieling, H. W. H. J. van Tits, J. A. van der Vliet, and R. J. A. Goris; 1993. Endotoxemia following major vascular operations. J. Vasc. Surg. 18:853-857. 
17. Damas, P., D. Ledoux, M. Nys, Y. Vrindts, D. De Groote, P. Franchimont, and M. Lamy; 1992. Cytokine serum level during severe sepsis in human IL-6 as a marker of severity. Ann. Surg. 215:356-362.
18. Frieling, J. T. M., M. Van Deuren, J. Wijdenes, J. W. M. Van der Meer, C. Clement, C. J. Van der Linden, and R. W. Sauerwein; 1995. Circulating interleukin-6 receptor in patients with sepsis syndrome. J. Infect. Dis. 171: 469-472. 
19. van Deuren, M., J. van der Ven-Jongekrijg, A. K. Bartelink, R. van Dalen, R. W. Sauerwein, and J. W. van der Meer; 1995. Correlation between proinflammatory cytokines and antiinflammatory mediators and the severity of disease in meningococcal infections. J. Infect. Dis. 172:433-439. 
20. Dokter, W. H. A., A. J. Dijkstra, S. B. Koopmans, B. K. Stulp, W. Keck, M. R. Halie, and E. Vellenga; 1994. G(Anh)MTetra, a natural bacterial cell wall breakdown product, induces interleukin-1beta and interleukin-6 expression in human monocytes. J. Biol. Chem. 269:4201-4206.
21. Mattsson, E., L. Verhage, J. Rollof, A. Fleer, J. Verhoef, and H. van Dijk; 1993. Peptidoglycan and teichoic acid from Staphylococcus epidermidis stimulate human monocytes to release tumour necrosis factor alpha-interleukin-1(beta) and interleukin-6. FEMS Immunol. Med. Microbiol. 7:281-288. 
22. Timmerman, C. P., E. Mattson, L. Martinez-Martinez, L. de Graaf, J. A. G. van Strijp, H. A. Verbrugh, J. Verhoef, and A. Fleer; 1993. Induction of tumor necrosis factor from human monocytes by staphylococci and staphylococcal peptidoglycans. Infect. Immun. 61:4167-4172.

Validity of interpretation criteria for standardized Western blots (immunoblots) for serodiagnosis of Lyme borreliosis based on sera collected throughout Europe.

Hauser U; Lehnert G; Wilske B.

ABSTRACT: Western blotting (WB; immunoblotting) is a widely used tool for the serodiagnosis of Lyme borreliosis (LB), but so far, no generally accepted criteria for performance and interpretation have been established in Europe. The current study was preceeded by a detailed analysis of WB with whole-cell lysates of three species of Borrelia burgdorferi sensu lato (U. Hauser, G. Lehnert, R. Lobentanzer, and B. Wilske, J. Clin. Microbiol. 35:1433-1444, 1997). In that study, interpretation criteria for a positive WB result were developed with the data for 330 serum samples (from patients with LB in different stages [n = 189] and from a control group [n = 141]) originating mostly from southern Germany. In the present work, the interpretation criteria for strains PKo (Borrelia afzelii) and PBi (Borrelia garinii) developed in the previous study were reevaluated with 224 serum samples (from patients with LB in different stages [n = 97] and from a control group [n = 127]) originating from throughout Europe that were provided by the European Union Concerted Action on Lyme Borreliosis (EUCALB). De novo criteria were developed on the basis of the reactivities of the EUCALB sera and were evaluated with the data for the samples from southern Germany. Comparison of all results led to the following recommendations:

• For WB for immunoglobulin G (IgG),
• for PKo at least two bands among
• p83/100,
• p58,
• p43,
• p39,
• p30,
• OspC,
• p21,
• p17, and
• p14 band
• and for PBi at least one band among
• p83/100,
• p39,
• p30,
• OspC,
• p21, and
• p17 band;
• for WB for IgM,
• for PKo at least one band among
• p39,
• OspC, and
• p17 or a strong p41 band
• and for PBi at least one band among
• p39, and
• OspC or a strong p41 band.

PMID: 10364592 UI: 99294763

J Clin Microbiol 2000 Jun;38(6):2097-2102.

A European Multicenter Study of Immunoblotting in Serodiagnosis of Lyme Borreliosis.

Robertson J; Guy E; Andrews N; Wilske B; Anda P; Granstrom M; Hauser U; Moosmann Y; Sambri V; Schellekens J; Stanek G; Gray J

[Record as supplied by publisher]
ABSTRACT: A European multicenter study of immunoblotting for the serodiagnosis of Lyme borreliosis showed considerable variation in results obtained from tests with a panel of 227 serum samples. Six laboratories used different immunoblot methods, and a wide range of bands was detected in all the assays. Multivariable logistic regression analysis of data from individual laboratories was used to determine the most discriminatory bands for reliable detection of antibodies to Borrelia burgdorferi sensu lato. These bands were used to construct individual interpretation rules for the immunoblots used in the six laboratories. Further analysis identified a subset of eight bands, which were important in all the laboratories, although with variations in significance. Possible European rules, all closely related, were formulated from these bands, although there was no single rule that gave high levels of sensitivity and specificity for all the laboratories. This is a reflection of the wide range of methodologies used, especially the use of different species and strains of B. burgdorferi sensu lato. The panel of European rules provides a framework for immunoblot interpretation which may be adapted in relation to the characteristics of Lyme borreliosis in local areas.

PMID: 10834959 UI: No Cit. ID assigned

J Chemother 1996 Feb;8 Suppl 2:83-90

Diffusion of 3-quaternary ammonium cephem antibiotics into cerebrospinal fluid of patients with bacterial meningitis.

Modai J

Cefepime and cefpirome are new beta-lactamase resistant parenteral cephalosporin derivatives whose spectrum of activity makes them suitable for use in the treatment of severe infections such as bacterial meningitis. However, the published information on the penetration of these new agents into human CSF and on their use in the treatment of bacterial meningitis are really scarce. Experimental studies have shown that cefepime and cefpirome penetrated remarkably well into the CSF of animals infected with Streptococcus pneumoniae, Streptococcus agalactiae, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Haemophilus influenzae type b or Pseudomonas aeruginosa. The mean changes in bacterial colony count in CSF after cefpirome or cefepime administration express the antibacterial activity of these drugs. Studies in patients show that cefepime and cefpirome crossed the blood-brain barrier and reached concentrations in the CSF that are bactericidal against most potential pathogens. Higher levels are likely to be achieved with multiple dosing and in the presence of inflamed meninges. No study has been performed to investigate the efficacy of cefpirome in the treatment of bacterial meningitis. Cefepime was as effective and safe as cefotaxime for treatment of patients with bacterial meningitis as shown in the only clinical trial.

Publication Types:

• Review
• Review literature

Tissue penetration of the fourth generation parenteral cephalosporins.

Wise R

In this review, the methods employed to study tissue penetration are discussed. Mathematical, animal and human models all have their positive and negative features, however studies in man must be central to studying the pharmacokinetics of these agents. The newly introduced parenteral cephalosporins (cefpirome, cefepime and cefoselis) have been studied in man. In general they penetrate tissues to similar extents to other beta-lactam agents. Non-specialised tissues such as inflammatory exudate and peritoneal fluid are rapidly and extensively penetrated. More specialised tissues (such as prostate) are penetrated to a moderate extent (30 to 50%). Certain sites - such as the CSF - are poorly penetrated (c. 5 to 10%). It is important to have a knowledge of this information prior to therapeutic trials of such agents.

Publication Types:

• Review
• Review, tutorial

Lyme Borreliosis in Rhesus Macaques: Effects of Corticosteroids on Spirochetal Load and Isotype Switching of Anti-Borrelia burgdorferi Antibody

Andrew R. Pachner¹, Kei Amemiya², Melanie Bartlett¹, Henry Schaefer², Kiran Reddy², and Wei-Fen Zhang¹

Mandell, Douglas and Bennett's Principles and Practice of Infectious Diseases

Book/CD-ROM Package 5/e
2 vol. set +CD-ROM,

Gerald L. Mandell (Editor), John E. Bennett (Editor), Raphael Dolin (Editor)

"... Lyme arthritis MAY usually be treated successfully with either oral or intravenous antibiotic therapy. In MOST cases, 30-day courses of oral doxycycline or amoxicillin or 2- to 4-week courses of intravenous ceftriaxone are adequate. In a decision analysis, oral therapy was preferable...and is considerably less expensive.... Treatment failures have occurred with any of the regimens given, and a second course of therapy MAY be necessary."

pg. 2145

"...It has been showen that virulent strains of Borrelia burgdorferi are able to resist elimination by phagocytic cells, thereby evading the first line in the host defense system against infection......Borrelia burgdorferi seems to cross a cell monolayer at intracellular junctions, although it can penetrate through the cytoplasm of a cell. In a rat model, permiability changes in the blood-brain barrier begin within 12 hours after inoculation with the spirochete, and the organism may be cultured from the cerebrospinal fluid within 24 hours..."

Journal of the American Medical Association (JAMA). 1992;267:1364-1367

Invasion of the Central Nervous System by Borrelia burgdorferi in Acute Disseminated Infection

Benjamin J. Luft, Raymond J. Dattwyler

........ In this prospective study of unselected patients, we found two thirds of the patients with disseminated infection had B burgdorferi DNA in their CSF. Although the presence of spirochetal DNA does not necessarily mean that viable B. burgdorferi were present in the CNS of these patients, this is very likely, given the fact that these patients had evidence of active clinical infection. The parameters have been used in the past to diagnose acute CNS infection may have greatly underestimated the true incidence of CNS involvement in this group of patients. Neither the clinical presentation nor routine laboratory tests accurately predicted which patients had B. burgdorferi DNA in their CSF.

....... Our findings demonstrate that B. burgdorferi can disseminate to the CNS very early on in the course of the infection with little or no clinical evidence of CNS invovement. Acute primary and secondary infections due to Treponema pallidum (syphilis) are also associated with a high rate of dissemination to the CNS......This study has important therapeutic implications as well. In the past, the recommended treatment of acute Lyme disease consisted of low doses of oral tetracycline or penicillin, even in patients with signs and symptoms of systemic and therefore potential meningeal involvement...an inordinately high failure rate....when ceftriaxone, an antibiotic that is highly active against B. burgdorferi, achieves high CSF levels, and has a prolonged half-life, was compared with penicillin for the treatment of late Lyme disease, ceftriaxone had a significantly higher success rate.

........ Among four patients with chronic Lyme arthritis, B. burgdorferi was found in the CSF of one patient with relapsing arthritis. This patient had no clinical evidence of CNS involvement and no intrathecal antibody production . This raises the possibility that the CNS may act as a sanctuary for B. burgdorferi, protecting it from the action of systemic anti-biotics and immunity and thereby allowing it to reseed the periphery intermittently.

This finding is especially important when considering the appropriate treatment of the chronic phase of this disease and whether the use of oral antibiotics alone, as suggested by some for chronic arthritis, is appropriate."

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