Transcript H. pylori

Another YCP controversy:
Membrane Potential influence with Clinical index and Bismuth Subcitrate Rol in some Chileans Study.
Flow Cytometry viability analisys and Real Clinical Impact from this new way to evaluate Bismuth
antibacterian actions.
• Correlation between Flow Cytometry in the evaluation of
Membrane Potential and Cellular Viability from HP
• And Actual Rol from Bismuth Subcitrate in relation to the
membrane equilibrium and Clinical Impact
Departamento de Microbiología
Facultad de Ciencias Biológicas
Universidad de Concepción
CITOMETRÍA DE FLUJO EN LA EVALUACIÓN DE
POTENCIAL DE MEMBRANA Y VIABILIDAD CELULAR
DE Helicobacter pylori.
Magíster T.M. Juan Luis Castillo
Navarrete
Título
Citometría de flujo en la evaluación de potencial de
membrana y viabilidad celular de Helicobacter pylori.
• Magister Juan Luis Castillo, Dra Apolinaria Garcia, Dr,
Fernando Kawaguchi, Dr Carlos Gonzalez, Dr Jaime
Madariaga, Sr, Ignacio Alfaro, Tamara Perez, Cinthya Perez.
• Departamento de Microbiología, Facultad de Ciencias
Biológicas, Universidad de Concepción.
• Laboratorio de Citometría de Flujo, Hospital del Trabajador
Concepción.
Helicobacter pylori
Patógeno gastroduodenal humano
Hábitat
pH óptimo
Mucosa gástrica
Agente etiológico:
6.0 - 7.0 (neutrófila)
Ureasa
•
•
•
•
Enfermedad péptica ulcerosa
Dispepsia no ulcerosa
Gastritis crónica superficial
Linfoma MALT
Crecimiento a pH < 4.5
Gastroenterology 1992: 102;720-727
Helicobacter pylori
Estómago
Capacidad de generar
potencial bioenergético
• Viabilidad
• Crecimiento
• Proliferación
Potencial de
membrana
Gradiente electroquímico de
protones (membrana interna)
Bacterias aeróbicas
• Egreso de protones
• Ingreso de electrones
Ingreso / Egreso de solutos
Interior negativo
-100 a -200 mV
Fuerza
protón motriz
Gradiente eléctrico
Potencial de
membrana
J Exp Biol 2000:203;51-59
Antimicrob Agents Chemother 2000:44;827-834
Cytometry 1999:35;55-63 / 2001:43;223-226
Potencial de
membrana
Disminución
Involucrado
Aumento
Depolarización
• Autolisis bacteriana
• Transporte de glucosa
• Quimiotaxis
Hiperpolarización
Se reduce a cero
Ionóforos
J Exp Biol 2000:203;51-59
Antimicrob Agents Chemother 2000:44;827-834
Cytometry 1999:35;55-63 / 2001:43;223-226
Impermeabilidad
membrana celular
Sustancias
fluorescentes / vitales
Indicador de
viabilidad celular
• Compuestos orgánicos con al menos 2 cargas (+)
• Compuestos cargados negativamente
Excluidos por membranas
celulares intactas
(eucariontes y procariontes)
Letter Appl Microbiol 2002:34;1-7
FEMS Microbiol Rev 2000:24;429-448
Antimicrob Agents Chemother 2000:44;827-834
Cytometry 1999:35;55-63 / 2001:43;223-226
Membrana Celular
Potencial de
membrana
Impermeabilidad de
la membrana a
ciertas sustancias
Indicador de estado
fisiológico de la
membrana
Viabilidad
celular
Letter Appl Microbiol 2002:34;1-7
FEMS Microbiol Rev 2000:24;429-448
Antimicrob Agents Chemother 2000:44;827-834
Cytometry 1999:35;55-63 / 2001:43;223-226
Permeabilidad
membrana celular
Indicador de
muerte celular
Compuestos fluorescentes
que se unen a ácidos
nucleicos
•
•
•
•
•
Propidium iodado (PI)
TO-PRO-3
TO-PRO-1
Sytox Green
Bromuro de etidio
PI
Viabilidad
celular
Letter Appl Microbiol 2002:34;1-7
FEMS Microbiol Rev 2000:24;429-448
Antimicrob Agents Chemother 2000:44;827-834
Cytometry 1999:35;55-63 / 2001:43;223-226
Potencial de
membrana
Eucariontes
Procariontes
Citometría de
Flujo
Bacterias en
crecimiento
activo
Microelectrodos
Cationes lipofílicos
radiomarcados
Valor promedio de
suspensión celular
completa
J Exp Biol 2000:203;51-59
Antimicrob Agents Chemother 2000:44;827-834
Cytometry 1999:35;55-63 / 2001:43;223-226
Compuestos para
ensayos Potenciométricos
DiOC2(3)
(488 nm)
Carbocianinas
(slow response probes)
•Fluorescencia verde:
• Se altera con el tamaño celular y
presencia de agregados.
• Es independiente del PM
• Fluorescencia roja:
• Dependiente del tamaño y del PM
•Razón F. roja / F. verde:
• Medida de PM
Independiente del tamaño
Potencial de Membrana: Indicador
confiable de muerte celular
DiOC2(3): (3,3´-dietiloxacarbocianina iodada
Antimicrob Agents Chemother 2000:44;827-834
Cytometry 1999:35;55-63 / 2001:43;223-226
Handbook of fluorescent probes, 2002. (www.probes.com)
Objetivos Generales
Determinar por citometría de flujo, los parámetros de
tamaño, forma celular, viabilidad celular y potencial
de membrana en bacterias.
Determinar el efecto que ejercen sobre el potencial de
membrana y la viabilidad celular de H. pylori, diversas
sustancias que tienen acción contra esta bacteria.
Objetivos específicos
Determinar, mediante citometría de flujo, los parámetros
de tamaño, forma, viabilidad celular y potencial de
membrana, en E. coli y S. aureus.
Determinar, mediante citometría de flujo, los parámetros
de tamaño, forma, viabilidad celular y potencial de
membrana, en H. pylori.
Evaluar el efecto de agentes antibacterianos usados
comúnmente en el tratamiento de erradicación de
H. pylori, sobre el potencial de membrana y viabilidad
celular de H. pylori.
Hipótesis
Mediante citometría de flujo, es posible determinar los
parámetros de tamaño, forma celular, potencial de
membrana y viabilidad celular en Helicobacter pylori.
Los diversos agentes antibacterianos utilizados en el
tratamiento de la infección por Helicobacter pylori, producen
alteraciones en la células bacteriana, tanto de tamaño,
morfología, como también en el potencial de membrana y
viabilidad celular; parámetros determinables mediante
citometría de flujo.
Estimación de bio
volumen bacteriano
DY en Gram positivos
y Gram negativos
Ácidos nucleicos y
viabilidad celular
Efecto de antimicrobianos
sobre bacterias.
Análisis
monoparamétrico de
poblaciones celulares
Cytometry 2001:44;188-94
Appl Environ Microbiol 1998:64;3900-9
Cytometry 2000:41;41-5
Cytometry 1999:35;55-63
Antimicrob Agents Chemother 2000:44;827-834
Methods 2000:21;271-279
Cytometry 2001:43;223-226
Antimicrob Agents Chemother 2000:44;682-687
J Microbiol Methods 2000:42(1);1-2
Letters Appl Microbiol 2002:34;1-7
Cytometry 1997:29;298-305
Cytometry 1994:17;302-309
Cytometry 1994:17;302-9
Antimicrob Agents Chemother 2000:44;676-681
Antimicrob Agents Chemother 1998:42;1195-1199
FEMS Microbiol Rev 2000:24;429-448
Antimicrob Agents Chemother 2000:44;682-687
Cytometry Part A 2003:53A;97-102
Cytometry 1998:32;241-254
Cytometry 2001:43;55-68
Cepas bacterianas
Escherichia coli ATCC 25922
Staphylococcus aureus ATCC 29213
Helicobacter pylori ATCC 43504
Escherichia coli K12
Pseudomona aeruginosa
Acinetobacter spp.
Bacillus subtilis
Helicobacter pylori 636-C, 594-C,
612-C, 632-C633-A
Viabilidad celular
Potencial de membrana
Eflujo a BrEt
Estimación de
patrones de tamaño
bacteriano
Cultivo líquido (18 hrs, 37°C) en caldo
Müller Hinton filtrado (0.20 mm)
Bacterias vivas
Bacterias muertas
Protocolo básico de trabajo
Cultivo líquido (18 hrs, 37°C)
Tubo
Células vivas (ml) Células muertas (ml)
1
15
0
2
12
3
3
9
6
4
6
9
5
3
12
6
0
15
Protocolo de evaluación de viabilidad
• 15 ml células vivas y/o muertas
• 1.0 ml de PBS
• 10 ml PI (1 mg/ml)
• Incubar 5 minutos a T° ambiente y en oscuridad
• Adquirir en Citómetro de flujo
• Analizar fluorescencia roja
Protocolo de evaluación de DY
• 15 ml células vivas y/o muertas
• 1.0 ml de PBS (o PBS-EDTA)
• 30 ml DiOC2(3) 1 mM (30 mM Concentración final)
• Incubar 4 minutos a T° ambiente y en oscuridad
• Adquirir en Citómetro de flujo
• Analizar fluorescencia roja y verde
Metodología
Citometría de Flujo
Equipo
• FACSCalibur, B.D.
• Láser de Argón, 488 nm.
Adquisición
• Software CellQuest v3.3
• Máximo de 1000 eventos/seg.
• 20.000 eventos celulares
Análisis
• Software CellQuest v.3.3
• Media de intensidad de fluorescencia
verde y roja.
• Cálculo razón F. roja / F. verde
• Software Flow Explorer
Rev Méd Chile 1999:127;1385-1397
Antimicrob Agents Chemother 2000:44;827-834
Cytometry 1999:35;55-63 / 2001:43;223-226
http://www.cyto.purdue.edu/flowcyt/software/flowex4_files/Install4.exe
Tubo
1PM.001
PM.001
(CELLQuest)
Email
Flow explorer
CELLQuest
Cáculo de
razón FL2/FL1
FACS Convert
Nueva fila
(1pm.001)
Gráficos
Mac
Email
Resultados
• Estimación patrones de tamaño
• Incorporación de PI en S. aureus y E. coli
• DY en S. aureus y E. coli
• Incorporación de PI y DY en H. pylori
• Eflujo a bromuro de etidio en S. aureus y E. coli
• Eflujo a bromuro de etidio en H. pylori
Definición de gate de análisis
Estimación de patrones de tamaño
Markers
(+/- channels)
M1
M2
M3
P. aeuriginosa
Acinetobacter sp.
M4
M5
M6
M7
M8
E. coli
B. subtilis
Estimación de patrones de tamaño
Estimación de patrones de tamaño en cepas de H. pylori
Resultados
• Estimación patrones de tamaño
• Incorporación de PI en S. aureus y E. coli
• DY en S. aureus y E. coli
• Incorporación de PI y DY en H. pylori
• Eflujo a bromuro de etidio en S. aureus y E. coli
• Eflujo a bromuro de etidio en H. pylori
Incorporación de PI
Células vivas
Células vivas y
muertas (1:1)
51.37%
0.98%
Células muertas
99.03%
E. coli:E.coli
Incorporación
dede PI
porcélulas
células
muertas por calor
: Incorporación
PI por
muertas
120,00
100,00
Porcentaje
R = 0.997
80,00
60,00
40,00
20,00
0,00
0,000
3,000
6,000
9,000
12,000
15,000
Células muertas x 10 6
S. aureus:
Incorporación
PIpor
por
células
muertas por calor
S. aureus:
Incorporaciónde
de PI
células
muertas
90,00
80,00
R = 0.692
Porcentaje
70,00
60,00
50,00
40,00
30,00
20,00
10,00
0,00
0,000
3,000
6,000
9,000
Células muertas x 10
12,000
4
15,000
Agregados y
destrucción
celular
E.coli : Incorporación
de por
PI por
células muertas
muertas (shock frío).
E. coli: Incorporación
de PI
células
(Shock frío). Ensayo por triplicado.
Ensayo en triplicado.
100,00
90,00
R = 0.978
80,00
Porcentaje
70,00
60,00
50,00
40,00
30,00
20,00
10,00
0,00
0,000
3,000
6,000
9,000
Células muertas x 10
12,000
15,000
6
S. aureus
: Incorporación
depor
PI por
célulasmuertas
muertas (shock frío).
S. aureus:
Incorporación
de PI
células
(Shock frío). Ensayo por triplicado.
Ensayo en triplicado.
100,00
90,00
R = 0.981
80,00
Porcentaje
70,00
60,00
50,00
40,00
30,00
20,00
10,00
0,00
0,000
3,000
6,000
9,000
Células muertas x 104
12,000
15,000
Resultados
• Estimación patrones de tamaño
• Incorporación de PI en S. aureus y E. coli
• DY en S. aureus y E. coli
• Incorporación de PI y DY en H. pylori
• Eflujo a bromuro de etidio en S. aureus y E. coli
• Eflujo a bromuro de etidio en H. pylori
Estimación de DY
dep.
pol.
Células vivas
dep.
dep.
pol.
Células vivas y muertas (1:1)
pol.
Células muertas
Estimación de DY
dep.
dep.
Flow
pol.
Explorer
pol.
Razón FL2/FL1
Células vivas y muertas (1:1)
S. aureus: Células polarizadas y depolarizadas.
Razón flourescencia roja / verde (DioC2(3)). Ensayo por triplicado.
100,00
R = - 0.982 (pol)
90,00
80,00
Porcentaje
70,00
60,00
50,00
40,00
30,00
20,00
R = 0.982 (dep)
10,00
0,00
0,000
3,000
6,000
9,000
12,000
15,000
Células muertas x 10 4
E. coli: Células polarizadas y depolarizadas.
Razón flourescencia roja / verde (DioC2(3)). Ensayo por triplicado.
100,00
90,00
R = - 0.993 (pol)
80,00
Porcentaje
70,00
60,00
50,00
40,00
30,00
20,00
R = 0.993 (dep)
10,00
0,00
0,000
3,000
6,000
9,000
Células muertas x 10
12,000
6
15,000
Resultados
• Estimación patrones de tamaño
• Incorporación de PI en S. aureus y E. coli
• DY en S. aureus y E. coli
• Incorporación de PI y DY en H. pylori
• Eflujo a bromuro de etidio en S. aureus y E. coli
• Eflujo a bromuro de etidio en H. pylori
H. pylori: Incorporación de PI
H. pylori: Incorporación de DioC2(3)
Resultados
• Estimación patrones de tamaño
• Incorporación de PI en S. aureus y E. coli
• DY en S. aureus y E. coli
• Incorporación de PI y DY en H. pylori
• Eflujo a bromuro de etidio en S. aureus y E. coli
• Eflujo a bromuro de etidio en H. pylori
Eflujo a Bromuro de Etidio
Ingreso por difusión pasiva
Intercalante
Monocatiónico
Sistema de eflujo del tipo MFS (Major facilitator superfamiliy):
• Dependiente de la fuerza protón motriz
• Sustratos:
• Fluoroquinolonas hidrofílicas
• Compuestos orgánicos monocatiónicos (Acriflavina,
BrEt, Bromuro de tetrafenilfosfonio)
Fluorescencia
Balance entre influjo y eflujo
Exclusión
Bajo influjo y/o presencia de eflujo
Protocolo de evaluación de eflujo
a Bromuro de Etidio
• 15 ml células vivas y/o muertas
• 1.0 ml de PBS
• 20 ml BrEt (20 mg/ml concentración final)
• Incubar 5 minutos a T° ambiente y en oscuridad
• Adquirir en Citómetro de flujo
• Analizar fluorescencia roja.
Cytometry 1994:17;302-309
Eflujo a Bromuro de Etidio
1.29 %
Células vivas
97.95 %
Células muertas
Incorporación de PI y eflujo a bromuro de etidio
E. coli
S. auerus
R=0.999
R=0.999
R=0.999
R=0.998
Resultados
• Estimación patrones de tamaño
• Incorporación de PI en S. aureus y E. coli
• DY en S. aureus y E. coli
• Incorporación de PI y DY en H. pylori
• Eflujo a bromuro de etidio en S. aureus y E. coli
• Eflujo a bromuro de etidio en H. pylori
H. pylori: Eflujo a bromuro de etidio
H. pylori: Incorporación de PI
H. pylori: Eflujo a BrEt
H. pylori: Incorporación de DioC2(3)
Discusión
• Desarrollo de método de estimación de
patrones de tamaño
• Incorporación de PI en E.coli y S.aureus
• Ensayo de DY en E.coli y S.aureus
• Uso de Razón de Fluorescencia Roja/verde
• H. pylori: PI, BrEt y DY
Discusión
Desarrollo de método de estimación de patrones de tamaño
1.
2.
3.
4.
Medición de FSC usada ampliamente
Indice de refracción
Aplicaciones
Limitaciones:
• Células en suspensión
• Tamaño no cuantificable
• Mezcla de poblaciones celulares
5. Ventajas:
• Método sencillo y económico
• Uso de parámetros intrínsecos a la células
Discusión
Incorporación de PI en E.coli y S.aureus
1.
2.
3.
4.
5.
Concepto
Controles
Linealidad
Definición de viabilidad celular
Ventajas:
• Método sencillo y económico
• Combinación con parámetros intrínsecos a la
células
Discusión
Ensayo de DY en E.coli y S.aureus
1. Metodología ampliamente establecida
2. Disponibilidad de inmunerables flurocromos
(http://probes.invitrogen.com/handbook/sections/2200.html)
3. Aplicaciones
4. Limitaciones:
• Células en suspensión y no aisladas
• Costo, infraestructura y softwares de apoyo
5. Ventajas:
• Método rápido
• Información sobre poblaciones celulares
• Posibilidad de cuantificar
Discusión
*
***
DIOC2(3)
Agregados de
DIOC2(3)
Fluorescencia verde
Fluorescencia roja
Razón Fluorescencia
Roja / verde
DY
- -* -
Uso de Razón de Fluorescencia Roja/verde
=
- ** *** ** *- -* *-
DY
- - - - * * * * * *- *** ***** **** *** *-*
* -* ** ** ** **- *- -*
<
<<<
=
Proporcional al tamaño
Sensible a presencia
de agregados
Sensible a DY e
independiente del tamaño
Discusión
H. pylori: PI, BrEt y DY
1. Barrera de permeabilidad
2. Sistema de eflujo (operón hefABC: Nº acceso
AF059041)
3. pH, medio ambiente y DY:
• Adecuación del DY
• DY positivo:
• Uso de oxonoles (DiBAC4(3)) (evidencias
células depolarizadas)
4. Características fisiológicas aún desconocidas
Conclusiones
1
Se desarrolla un método de análisis que permite la
estimación de patrones de tamaño celular de células
bacterianas por citometría de flujo.
2
En E. coli y S. aureus, la citometría de flujo evalúa
exitosamente viabilidad celular mediante la
incorporación de yoduro de propidio, DY, usando
DIOC2(3) y la presencia de eflujo a
bromuro de etidio.
Conclusiones
3
En E. coli y S. aureus, la evaluación por citometría de
flujo de la incorporación de yoduro de propidio, la
estimación de DY y la presencia de eflujo a bromuro
de etidio, en conjunto se pueden considerar criterios
para definir viabilidad celular
4
En H. pylori, bajo las condiciones experimentales
ensayadas no fue factible evaluar viabilidad celular,
definida por la incorporación de yoduro de propidio,
DY, usando DIOC2(3) ni evaluar la presencia de
eflujo a bromuro de etidio.
Proyecciones
Patrones
de tamaño celular
Estudio de acción de diversas
sustancias sobre bacterias
Incorporación de PI
Viabilidad v/s cultivabilidad
Estimación de DY
Eflujo a BrEt
Búsqueda de otras condiciones
metodológicas en H. pylori
Proyecciones
Este trabajo sienta las bases para el desarrollo de una línea de trabajo
relacionada con la presencia y actividad de sistemas de eflujo en bacterias,
desde un punto de vista fisiológico y funcional.
Estudio de sistemas de eflujo y resistencia a drogas en células eucariontes,
en especial, células neoplásicas y el fenotipo MDR (Multi Drug Resistant),
particularmente la presencia y actividad de eflujo de la glicoproteína p
(DIOC2(3) es sustrato de la glicoproteína p).
El estudio integral de viabilidad celular en E. coli y S. aureus, es hasta
donde se tiene conocimiento, pionero en nuestro país, por lo que debiera
fomentar el uso de la citometría de flujo en estudios de microbiológicos y en
especial bacteriológicos.
• Similar morphologic and outer membrane
changes were observed following growth in ironlimiting medium and at the MICCBS that
inhibited the growth of all three strains.
• These changes, which were also observed for
iron-limited bacteria, were alleviated by the
addition of iron to the cultures.
• H. pylori ATP levels, reduced in iron-limiting medium,
were below the limits of detection in two of the three
strains following exposure to bismuth.
• The addition of iron partially restored bacterial ATP
levels in these two strains, although not to normal
concentrations.
• In contrast, exposure of the same strains to the MICCBS
failed to deplete intracellular levels of iron, which were
significantly reduced by culturing in iron-limiting
medium.
• Thus, the antimicrobial effect of bismuth and of iron limitation on H.
pylori may be similar.
• However, the respective mechanisms of intracellular action would
appear to be mediated by different pathways within the cell.
• Prior to the discovery of the link between Helicobacter pylori and
peptic ulcer disease (10), ulcers were thought to result from
abnormal levels of gastric acid secretion.
• Bismuth compounds, commonly prescribed for ulcers, were initially
thought to act as a barrier to the digestive effects of stomach acid by
coating the ulcer site (31).
• Instead, it appears that H. pylori is highly susceptible to bismuth
compounds (39), and treatment with colloidal bismuth subcitrate
(CBS) is associated with a reduction in bacterial numbers and a
concurrent reduction in gastritis in vivo (37).
• Despite these findings, bismuth monotherapy often fails
to completely eradicate these bacteria (13, 43), an
observation that correlates with increasing ulcer relapse
rates over time (34).
• The oldest regimen, an extremely effective one for
clearing H. pylori infection, uses bismuth in combination
with certain antibiotics (2, 51).
• Bismuth “triple therapy” consists of a bismuth compound
(usually CBS or bismuth subsalicylate) in combination
with metronidazole and tetracycline or amoxicillin (16)
and reportedly cures 87.9% of patients within 1 week of
treatment and 89.2% of patients within 2 weeks of
treatment (49)
• H. pylori strains resistant to bismuth have not been reported and
presumably arise at a lower frequency than strains resistant to
antimicrobial agents such as nitroimidazoles, macrolides, and
tetracycline (40).
•
Bismuth compounds may reduce the development of resistance to
coadministered antibiotics (27) and are also effective at treating H.
pylori strains with established resistance to other antibiotics (3, 40).
• How bismuth is toxic to H. pylori is not known.
• A number of studies have linked the antimicrobial activities of many
heavy metals, including bismuth, to their effects on iron uptake by
bacteria (1, 21, 23, 24).
• Iron is required for growth because it is a cofactor for many essential
enzymatic processes (42), and many of the observed effects of bismuth
on bacteria could be the result of iron limitation.
• These effects include a reduction in intracellular ATP levels (44),
inhibition of protein and cell wall synthesis because loss Sodio/Potasio
Bumb equilibrium of membrane potential function (32) with reduction
in capsular polysaccharide.
•
Prior to the discovery of the link between Helicobacter pylori and peptic ulcer
disease (10), ulcers were thought to result from abnormal levels of gastric acid
secretion.
• Bismuth compounds, commonly prescribed for ulcers, were initially thought to act
as a barrier to the digestive effects of stomach acid by coating the ulcer site (31).
• Instead, it appears that H. pylori is highly susceptible to bismuth compounds (39),
and treatment with colloidal bismuth subcitrate (CBS) is associated with a
reduction in bacterial numbers and a concurrent reduction in gastritis in vivo (37).
• Despite these findings, bismuth monotherapy often fails to completely eradicate
these bacteria (13, 43), an observation that correlates with increasing ulcer relapse
rates over time (34).
• The oldest regimen, an extremely effective one for clearing H. pylori infection, uses
bismuth in combination with certain antibiotics (2, 51).
• Bismuth “triple therapy” consists of a bismuth compound (usually CBS or bismuth
subsalicylate) in combination with metronidazole and tetracycline or amoxicillin
(16) and reportedly cures 87.9% of patients within 1 week of treatment and 89.2%
of patients within 2 weeks of treatment (49).
• H. pylori strains resistant to bismuth have not been reported and presumably arise a
lower frequency than strains resistant to antimicrobial agents such as nitroimidazol
macrolides, and tetracycline (40).
• Bismuth compounds may reduce the development of resistance to coadministered
antibiotics (27) and are also effective at treating H. pylori strains with established
resistance to other antibiotics (3, 40).
• Even this mechanism is clear, habitually gastroenterologists don’t know how bismut
toxic to H. pylori is not known.
• A number of studies have linked the antimicrobial activities of many heavy metals,
including bismuth, to their effects on iron uptake by bacteria (1, 21, 23, 24).
• Iron is required for growth because it is a cofactor for many essential enzymatic
processes (42), and many of the observed effects of bismuth on bacteria could be the
result of iron limitation.
•
These effects include a reduction in intracellular ATP levels (44), inhibition of prot
and cell wall synthesis and of membrane function (32), and a reduction in capsular
polysaccharide production (19, 20, 22, 28).
• A reduction in outer membrane lipopolysaccharide (LPS) expression
occurs when H. pylori is deprived of iron (J. Keenan, unpublished
data). Castillo’s group already demonstrated through flow cytometry,
celular form, size, membrane potential and celular viability. Again,
Several iron-repressible outer membrane proteins are concomitantly
up-regulated (18, 29, 52).
• These observations led us to hypothesize that the antimicrobial action
of bismuth could be due to it competitively inhibiting iron uptake.
• To test this hypothesis, we searched for indicators of iron limitation
in bismuth-exposed H. pylori and investigated whether iron could
protect these bacteria from the antimicrobial effect of bismuth.
•
MATERIALS AND METHODS
•
Bacterial strains and cultures. Three well-characterized H. pylori strains
were used in this study: H. pylori 60190 (ATCC 49503), a cag pathogenicity
island (PAI)-positive toxigenic strain (5, 14); cag PAI-negative H. pylori
strain Tx-30a (ATCC 51932) (14), which fails to produce detectable
cytotoxin activity in vitro (35); and mouse-adapted H. pylori Sydney strain 1
(SS1) (33).
Individual strains were grown in the base medium, brucella broth (BB;
Difco, Detroit, Mich.) supplemented with 5% fetal bovine serum (FBS;
Gibco BRL, Auckland, New Zealand), for 72 h at 37°C under
microaerophilic conditions and with constant rotation (120 rpm).
•
•
Iron limitation and bismuth inhibition were achieved by growing the strains
in BB-5% FBS with the addition of an iron chelator (deferoxamine
mesylate; Sigma, St. Louis, Mo.) at a final concentration of 50 μM or CBS at
the MIC (MICCBS) determined for each strain (see below).
•
Iron salts (ferrous ammonium sulfate; Sigma) at final concentrations of 50
to 1,000 μM were added to the base medium containing MICCBS to
determine the amount of iron required to alleviate the effects of bismuth.
•
The deferoxamine, CBS, and iron salts solutions were prepared immediately
before use. Deferoxamine and iron salts solutions were filter sterilized prior
to addition to the medium.Morphologic changes.
•
The morphologic changes in the bacteria were assessed by transmission
electron microscopy (TEM) over the 72-h r period. Washed bacteria were
placed on carbon-colloidin-coated mesh grids and negatively stained with
1% aqueous phosphotungstic acid (pH 7.0).
•
Photographs were taken by using a CM12 transmission electron microscope
(Philips).Determining MICCBS values for individual H. pylori strains.
•
MICCBS values were determined by using 12-well tissue culture plates
inoculated with CBS stock solution to a final volume of 2 ml/well.
•
CBS, prepared from bismuth citrate powder and ammonia (26), was
combined with 2 × 107 H. pylori organisms per ml at final concentrations of
0, 1, 2, 4, 8, 16, 32, and 64 mg/liter in the base medium, and the mixture was
incubated for 72 h in a microaerophilic environment with constant rotation.
• H. pylori growth assessed by measuring the optical density at 650 nm
in each well (corrected by using uninoculated controls) was
confirmed as the growth of gram-negative, urease-positive colonies
on blood agar plates.
• Each strain was tested six times per experiment, performed on three
separate occasions, and controls containing ammonia without
bismuth were included to ensure that any inhibitory effects were due
to bismuth alone.OMV.
•
Bacteria were removed from 72-h broth cultures by centrifugation
(10,000 × g, 15 min, 4°C), and the supernatants were
ultracentrifuged (100,000 × g, 2 h, 4°C) to recover outer membrane
vesicles (OMV) as described previously (30).
• The OMV pellet was washed twice with phosphate-buffered saline
(PBS) and assayed for protein content (36). OMV components
separated by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis under reducing conditions were silver stained to
visualize protein and LPS profiles (30).
•
OMV-associated protease activity was detected by zymography (29).
•
Briefly, OMV were electrophoresed under nonreducing conditions through
an acrylamide gel containing a copolymerized substrate (gelatin).
•
After extensive washing to remove sodium dodecyl sulfate, the gel was
incubated in 50 mM Tris buffer (pH 7.4) with 10 mM calcium chloride for 4
h at 37°C.
•
OMV-associated proteolytic activity was visualized as clear bands (indicative
of substrate lysis) against the blue background of the gels following
Coomassie blue staining.Collection of samples for analysis of ATP and
intracellular iron levels.
•
H. pylori organisms were grown in BB-5% FBS for 24 h before being
adjusted to the conditions described above for the remaining 48 h of
culturing.
•
At 72 h, a 1-ml aliquot was removed to determine the effects of the various
culture conditions on bacterial numbers by viable colony counting. A second
aliquot was snap-frozen (in liquid nitrogen) and stored at −80°C prior to
analysis of ATP levels.
•
The remaining bacteria were harvested (10,000 × g, 20 min) to determine
intracellular iron levels. ATP and iron levels were determined in relation to
bacterial dry weight.Viable colony counting.
•
After serial dilution in PBS, bacterial titers were inferred from CFU per
milliliter, determined by spreading bacterial suspensions over the surface of
blood agar plates containing 5% defibrinated sheep blood.
•
The agar plates were incubated for 5 days at 37°C under microaerophilic
conditions (as described above).Analysis of ATP levels.
• ATP levels were measured by using a luminescent ATP detection
assay kit (ATPLite-Packard Bioscience) according to manufacturer
directions.
• Briefly, frozen samples were quickly defrosted, and 100-μl portions
of the samples were added to the wells of 96-well tissue culture plates
in duplicate.
•
Following the addition of 50 μl of lysis solution, the plates were
shaken for 5 min (700 rpm), and then 50 μl of substrate buffer
solution was added.
• The plates were shaken for a further 5 min and dark adapted for 10
min before luminescence was measured with a luminometer (BMG
Labtechnologies).
• ATP standards, prepared for each of the four conditions used to
culture H. pylori and ranging from 10−5 M to the blank, were used
to generate a standard curve (Graphpad-Instat) from which
unknown sample concentrations were calculated.Analysis of iron
levels.
•
PBS-washed bacterial pellets were oven dried at 80°C for approximately 18 h in 1.5ml Eppendorf tubes and then transferred to 10-ml acid-washed flasks for the
determination of dry weights.
•
Each sample was dissolved in 1 ml of concentrated nitric acid at 55 to 65°C overnight.
•
Dissolved samples were cooled to an ambient temperature and then brought to 10 ml
with Milli-Q distilled water.
•
Samples were examined on a Spectra-10 (Varian) atomic absorption spectrometer, and
iron concentrations were determined from a standard curve that ranged from 0 to
5,000 μg/liter (25).
•
Statistical analyses. The effects of each of the growth conditions on bacterial iron and
ATP levels were evaluated by using a factorial analysis of variance.
•
Significant effects indicated by this analysis were further explored by using Fisher's
least-significant-difference test.
•
The ATP measurements were natural logarithm transformed prior to analysis, so that
very small values could be easily analyzed.
• RESULTS
•
•
•
•
•
•
•
Bismuth or iron limitation induces a change in H. pylori morphology and
outer membrane composition.
The MICCBS values were found to be 4 mg/liter (10 μM) for strains 60190
and SS1 and 8 mg/liter (20 μM) for Tx-30a.
The addition of ammonia alone (see Materials and Methods) did not inhibit
the growth of any of the strains. Thus, the inhibitory effects of CBS were due
to bismuth alone (results not shown).
Each of the three H. pylori strains was examined by TEM following 72 h of
growth in base medium (control), iron-limiting medium (containing 50 μM
deferoxamine), or medium containing MICCBS (Fig. 1).
Both iron limitation and exposure to MICCBS resulted in the transition of
H. pylori from helical to coccoid morphology (Fig. 1A); a larger proportion
of MICCBS-exposed bacteria had completed this transition by 72 h.
Exposure to MICCBS resulted in some more distorted forms of H. pylori
(Fig. 1B).
In contrast, bacteria grown under normal culture conditions exhibited
normal curved-rod morphology.
•
•
RESULTS
Bismuth or iron limitation induces a change in H. pylori morphology and outer membrane composition.
•
The MICCBS values were found to be 4 mg/liter (10 μM) for strains 60190 and SS1 and 8 mg/liter (20 μM) for Tx30a.
•
The addition of ammonia alone (see Materials and Methods) did not inhibit the growth of any of the strains.
•
Thus, the inhibitory effects of CBS were due to bismuth alone (results not shown).
•
Each of the three H. pylori strains was examined by TEM following 72 h of growth in base medium (control), ironlimiting medium (containing 50 μM deferoxamine), or medium containing MICCBS (Fig. 1).
•
•
Both iron limitation and exposure to MICCBS resulted in the transition of H. pylori from helical to coccoid
morphology (Fig. 1A)
A larger proportion of MICCBS-exposed bacteria had completed this transition by 72 h. Exposure to MICCBS
resulted in some more distorted forms of H. pylori (Fig. 1B).
•
In contrast, bacteria grown under normal culture conditions exhibited normal curved-rod morphology
•
There was little observable difference in the outer membrane proteins of individual
•
H. pylori strains following iron limitation or exposure to bismuth (Fig. 2).
•
Specific markers for iron stress were difficult to identify, but a 12-kDa protein band was clearly enhanced in the
presence of MICCBS or 50 μM deferoxamine (Fig. 2)
•
. Both iron-limited and MICCBS-exposed bacteria also had reduced levels of OMV-associated LPS (Fig. 3).
•
In addition, zymography revealed OMV-associated protease activity in each strain following growth in the presence
of MICCBS or under iron-limiting conditions (Fig. 4).
•
Iron protects H. pylori from the inhibitory effects of bismuth.
•
The addition of 250 μM iron protected strains 60190 and SS1 from the inhibitory effects of 10 μM
CBS. H. pylori Tx-30a, which required twofold more bismuth for growth inhibition (20 μM), also
required more iron (500 μM) to achieve any protective effect.
•
However, this amount of iron only reduced the bactericidal effect of bismuth on Tx-30a, as
indicated by growth after culturing on blood agar.
•
It did not completely reverse growth inhibition, as demonstrated by a slight increase in
absorbance readings after 72 h of culturing.
•
H. pylori grown in medium containing MICCBS and protective iron maintained the normal
curved-rod morphology typical of these bacteria (Fig. 1D).
•
Furthermore, this treatment of H. pylori 60190 (results not shown) and SS1 (Fig. 2) resulted in
outer membrane protein profiles similar to those of bacteria grown in base medium.
•
The provision of protective iron to H. pylori Tx-30a apparently failed to decrease the expression of
the 12-kDa protein band (Fig. 2B).
•
We speculate that this result may have been due to the visible overloading of the corresponding
lane.
•
However, it is possible that this protein is still expressed at a higher level in this strain under these
conditions.
•
Protease activity was no longer detectable in bacteria of all three strains grown in medium
containing MICCBS and protective iron (Fig. 4)
•
ATP but not iron levels are reduced following exposure of H. pylori to bismuth.
•
Intracellular iron levels of H. pylori exposed to MICCBS were compared with those of bacteria
cultured under normal and iron-limiting conditions to assess whether bismuth inhibited iron
uptake.
To achieve the biomass required for this analysis, cultures were grown overnight before being
exposed to bismuth or limiting iron for 48 h.
•
•
Colony counts confirmed that the established MICCBS values were sufficient to prevent growth
even at these much higher concentrations of bacteria, and inhibition was still overcome by the
addition of iron at protective concentrations (results not shown).
•
PBS used to wash the bacterial pellet was assayed for iron by atomic absorption spectrometry.
•
After three washes, the amount of iron present in the PBS supernatant was below the detectable
limits of the assay, indicating this number of washes to be sufficient for removing excess unbound
iron (results not shown).
•
H. pylori strains 60190 and SS1 grown in the presence of MICCBS had intracellular levels of iron
similar to those of bacteria cultured under normal conditions, as measured by atomic absorption
spectrometry.
•
In contrast, iron levels were significantly reduced following culturing in 50 μM deferoxamine
(Fig. 5).
•
However, MICCBS had an effect similar to that of iron limitation on the intracellular iron levels of
H. pylori Tx-30a.
•
All three strains cultured in the presence of MICCBS and protective iron had levels of
intracellular iron that were higher than those of controls but not significantly different from those
of bacteria cultured with protective iron alone.Bacterial
•
ATP levels were only significantly reduced in strains SS1 and 60190 following growth in ironlimiting medium (Fig. 6).
Exposure to MICCBS resulted in a reduction in ATP production to below the detectable limits of
the assay for these two strains, whereas ATP levels in H. pylori Tx-30a were not significantly
different from those in controls.
•
•
•
The addition of iron to bacterial cultures containing MICCBS increased bacterial ATP
concentrations in all three strains.
However, protective iron failed to restore H. pylori 60190 ATP levels to those observed following
culturing under normal or even iron-limiting conditions
•
RESULTS
•
Bismuth or iron limitation induces a change in H. pylori morphology and outer membrane composition.
•
The MICCBS values were found to be 4 mg/liter (10 μM) for strains 60190 and SS1 and 8 mg/liter (20 μM) for Tx30a.
•
The addition of ammonia alone (see Materials and Methods) did not inhibit the growth of any of the strains.
•
Thus, the inhibitory effects of CBS were due to bismuth alone (results not shown).
•
Each of the three H. pylori strains was examined by TEM following 72 h of growth in base medium (control), ironlimiting medium (containing 50 μM deferoxamine), or medium containing MICCBS (Fig. 1).
•
Both iron limitation and exposure to MICCBS resulted in the transition of H. pylori from helical to coccoid
morphology (Fig. 1A); a larger proportion of MICCBS-exposed bacteria had completed this transition by 72 h.
Exposure to MICCBS resulted in some more distorted forms of H. pylori (Fig. 1B).
•
In contrast, bacteria grown under normal culture conditions exhibited normal curved-rod morphology.There was
little observable difference in the outer membrane proteins of individual H. pylori strains following iron limitation
or exposure to bismuth (Fig. 2).
•
Specific markers for iron stress were difficult to identify, but a 12-kDa protein band was clearly enhanced in the
presence of MICCBS or 50 μM deferoxamine (Fig. 2).
•
Both iron-limited and MICCBS-exposed bacteria also had reduced levels of OMV-associated LPS (Fig. 3). In
addition, zymography revealed OMV-associated protease activity in each strain following growth in the presence of
MICCBS or under iron-limiting conditions (Fig. 4)
•
.Iron protects H. pylori from the inhibitory effects of bismuth.
•
The addition of 250 μM iron protected strains 60190 and SS1 from the inhibitory effects of 10 μM CBS. H. pylori
Tx-30a, which required twofold more bismuth for growth inhibition (20 μM), also required more iron (500 μM) to
achieve any protective effect.
•
However, this amount of iron only reduced the bactericidal effect of bismuth on Tx-30a, as indicated by growth
after culturing on blood agar.
•
It did not completely reverse growth inhibition, as demonstrated by a slight increase in absorbance readings after
72 h of culturing.
•
H. pylori grown in medium containing MICCBS and protective iron maintained the normal curved-rod
morphology typical of these bacteria (Fig. 1D).
•
Furthermore, this treatment of H. pylori 60190 (results not shown) and SS1 (Fig. 2) resulted in outer membrane
protein profiles similar to those of bacteria grown in base medium.
•
The provision of protective iron to H. pylori Tx-30a apparently failed to decrease the expression of the 12-kDa
protein band (Fig. 2B).
•
We speculate that this result may have been due to the visible overloading of the corresponding lane.
•
However, it is possible that this protein is still expressed at a higher level in this strain under these conditions
•
. Protease activity was no longer detectable in bacteria of all three strains grown in medium containing MICCBS
and protective iron (Fig. 4)
•
ATP but not iron levels are reduced following exposure of H. pylori to bismuth.
•
Intracellular iron levels of H. pylori exposed to MICCBS were compared with those of bacteria cultured under normal and
iron-limiting conditions to assess whether bismuth inhibited iron uptake.
•
To achieve the biomass required for this analysis, cultures were grown overnight before being exposed to bismuth or
limiting iron for 48 h.
•
Colony counts confirmed that the established MICCBS values were sufficient to prevent growth even at these much higher
concentrations of bacteria, and inhibition was still overcome by the addition of iron at protective concentrations (results
not shown)
•
. PBS used to wash the bacterial pellet was assayed for iron by atomic absorption spectrometry.
•
After three washes, the amount of iron present in the PBS supernatant was below the detectable limits of the assay,
indicating this number of washes to be sufficient for removing excess unbound iron (results not shown).
•
H. pylori strains 60190 and SS1 grown in the presence of MICCBS had intracellular levels of iron similar to those of
bacteria cultured under normal conditions, as measured by atomic absorption spectrometry. In contrast, iron levels were
significantly reduced following culturing in 50 μM deferoxamine (Fig. 5).
•
However, MICCBS had an effect similar to that of iron limitation on the intracellular iron levels of H. pylori Tx-30a.
•
All three strains cultured in the presence of MICCBS and protective iron had levels of intracellular iron that were higher
than those of controls but not significantly different from those of bacteria cultured with protective iron alone.Bacterial
ATP levels were only significantly reduced in strains SS1 and 60190 following growth in iron-limiting medium (Fig. 6).
•
Exposure to MICCBS resulted in a reduction in ATP production to below the detectable limits of the assay for these two
strains, whereas ATP levels in H. pylori Tx-30a were not significantly different from those in controls.
•
•
The addition of iron to bacterial cultures containing MICCBS increased bacterial ATP concentrations in all three strains.
However, protective iron failed to restore H. pylori 60190 ATP levels to those observed following culturing under normal
or even iron-limiting conditions
•
DISCUSSION
•
In this study, we have shown that exposure to MICCBS has the same effect on H. pylori as iron limitation during growth. H. pylori
cultured in the presence of MICCBS underwent a morphologic conversion from the bacillary to the coccoid form that was associated with
changes in outer membrane composition.
•
These changes, which were also observed in iron-limited bacteria, were prevented by supplementation of the medium with iron.
•
However, our hypothesis that the antimicrobial action of bismuth was due to the inhibition of iron uptake was challenged by unchanged
intracellular iron levels in two of the three H. pylori strains following culturing in the presence of this bismuth compound.Bismuthinduced changes in H. pylori morphology have been seen before (11, 45, 47), but our observations of accompanying changes in outer
membrane protein and LPS profiles are new.
•
The bismuth-induced changes included the up-regulation of a 12-kDa protein and two proteases and diminished LPS expression and are
identical to those observed in other studies in which H. pylori was grown under iron-limiting conditions (9, 29).
•
Furthermore, the addition of iron during growth mostly prevented these bismuth-induced changes. One possible exception was the
continued expression of a 12-kDa protein by H. pylori Tx-30a grown in the presence of MICCBS and protective iron, even though TEM
revealed helical forms of bacteria under these conditions.
•
This observation may be linked to the incomplete reversal of bismuth-induced growth inhibition in the presence of protective iron.
However, the apparent overloading of the corresponding lane may also be a contributing factor.
The change from helical to coccoid morphology was more rapid in the bismuth-exposed bacteria and was associated with a significant
decrease in viability. ATP levels were below the limits of detection in H. pylori strains 60190 and SS1 following exposure to bismuth,
whereas strain Tx-30a had ATP levels not significantly different from those seen under normal culture conditions, despite a rapid
conversion to the coccoid form that was likewise associated with decreased viability.
•
•
Strain Tx-30a was also associated, again in contrast to the other two strains, with a significant reduction in intracellular iron levels
following exposure to MICCBS. Both of these results would be consistent with a loss of membrane integrity (44), leading to an
accumulation of nonutilizable ATP in the extracellular medium and a concurrent loss of iron from the cell.
•
We speculate that this effect may reflect the higher bismuth concentration needed to inhibit this strain and may also be linked to the
incomplete reversal of bismuth-induced growth inhibition in the presence of protective iron.H. pylori 60190 and SS1 cultured with
MICCBS and protective iron had ATP levels higher than those found in the presence of bismuth alone but significantly lower than those
found under normal culture conditions.
•
•
The use of protective iron and MICCBS was also associated with an increase in intracellular iron levels and partial protection from bismuthinduced growth inhibition.
•
These observations, which would suggest that bismuth and iron were competing for uptake via the same pathway in these bacteria, were
further supported by our observation that iron limitation had a similar (but less severe) effect on H. pylori ATP levels and viability.
•
Unexpectedly, however, intracellular iron levels remained constant in these two strains following exposure to bismuth, whereas iron
limitation over the same growth period resulted in the depletion of iron.
•
These results would suggest that the mechanism for the antimicrobial effect of bismuth on these and other bacteria is not simply iron
starvation, as suggested previously (21).
•
Instead, the effect of bismuth only mimics the effect of iron starvation in H. pylori.ATP synthesis in bacteria occurs at the cytoplasmic
membrane through the action of multisubunit enzymes (F1F0-ATPases) that utilize the membrane potential and electrochemical gradient
generated by respiration (48).
•
In H. pylori, inhibition of these enzymes is thought to lead to ATP depletion and the associated inhibition of other pathways that are
important for H. pylori survival (38).
•
Other potential targets for the inhibition of ATP synthesis include the iron-sulfur clusters and cysteine-containing heme groups that act as
electron carriers in the respiratory chain. The observation that bismuth complexes localize in the periplasmic space (between the cytoplasmic
and outer membranes) in H. pylori (4, 32) would be consistent with a mechanism of action of bismuth that may involve the inhibition of ATP
synthesis via one or more of these pathways
•
The accumulation of bismuth at the cytoplasmic membrane might have prevented iron incorporation into iron-utilizing proteins, thereby
creating a functional iron limitation within the respiratory chain. Thus, the bismuth-induced production of iron-repressible outer membrane
proteins could be a response to perceived iron stress, despite no change in intracellular iron levels, an effect similar to that caused by other
heavy metals through competition for incorporation into iron binding proteins (8).
•
However, bismuth is also able to inhibit the electron transport chain through binding to the sulfhydryl groups within the enzyme complex, and
this mechanism might instead account for the rapid reduction in intracellular ATP levels (6, 7).
•
Interestingly, McGowan et al. showed that F1F0-ATPase activity is required for the survival of H. pylori when the external pH is nearly
neutral (38).
•
•
This finding leads us to speculate that the increased efficacy of quadruple therapy, in
which bismuth and a proton pump inhibitor are given in combination with two
antibiotics (17), is likely to be linked to this F1F0-ATPase-dependent survival at a
neutral pH.Despite these subtle differences in the mechanisms for shutting down ATP
synthesis, the effects are likely to be the same.
•
Moreover, the structural and compositional changes that we observed in the H. pylori
outer membrane are likely to have direct consequences on the survival of these
bacteria in the gastric mucosa. Reduced LPS synthesis could affect the stability of the
bacterial glycocalyx (46) as well as increase the exposure of surface antigens, thereby
rendering H. pylori more susceptible to host defenses and/or hydrophilic
antimicrobial agents (50)
•
.Interestingly, considerable strain-dependent variations in the ability to preserve ATP levels were observed
following culturing of the H. pylori strains in the presence of MICCBS and protective iron.
•
H. pylori 60190 ATP levels were considerably lower than those observed under both normal and iron-limiting
culture conditions, and one explanation might be an increased requirement for energy by this strain. H. pylori
60190 carries the complete cag PAI, a locus of about 37 kb containing up to 31 genes (12).
•
Several of these genes code for a type IV secretion system (41) that includes an ATP-regulated inner membrane
pore (53).
•
Supporting this hypothesis is the observation of higher levels of ATP in the other two strains grown under the
same conditions. These strains do not possess the cag PAI (Tx-30a) or carry only a partial cag PAI (SS1) (14, 15).
•
In summary, our results suggest that whereas the antimicrobial effects of bismuth and iron deprivation on H.
pylori may be similar, their respective mechanisms of intracellular action would appear to be mediated by different
pathways within the cell.
REFERENCES
1.al-Aoukaty, A., V. D. Appanna, and H. Falter. 1992. Gallium toxicity and adaptation in Pseudomonas fluorescens. FEMS Microbiol. Lett. 71:265-272.
2.Alarcon, T., D. Domingo, and M. Lopez-Brea. 1999. Antibiotic resistance problems with Helicobacter pylori. Int. J. Antimicrob. Agents 12:19-26. [PubMed] [Full
Text].
3.Andersen, L. P., H. Colding, and J. E. Kristiansen. 2000. Potentiation of the action of metronidazole on Helicobacter pylori by omeprazole and bismuth subcitrate. Int.
J. Antimicrob. Agents 14:231-234. [PubMed] [Full Text].
4.Armstrong, J. A., S. H. Wee, C. S. Goodwin, and D. H. Wilson. 1987. Response of Campylobacter pyloridis to antibiotics, bismuth and an acid-reducing agent in vitro—
an ultrastructural study. J. Med. Microbiol. 24:343-350. [PubMed].
5.Atherton, J. C., P. Cao, R. M. Peek, Jr., M. K. Tummuru, M. J. Blaser, and T. L. Cover. 1995. Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori.
Association of specific vacA types with cytotoxin production and peptic ulceration. J. Biol. Chem. 270:17771-17777. [PubMed] [Free Full Text
6.Baer, W., H. Koopmann, and S. Wagner. 1993. Effects of substances inhibiting or uncoupling respiratory-chain phosphorylation of Helicobacter pylori. Zentbl.
Bakteriol. 280:253-258.
7.Beil, W., C. Birkholz, S. Wagner, and K. F. Sewing. 1995. Bismuth subcitrate and omeprazole inhibit Helicobacter pylori F1-ATPase. Pharmacology 50:333-337.
[PubMed].
8.Bereswill, S., S. Greiner, A. H. van Vliet, B. Waidner, F. Fassbinder, E. Schiltz, J. G. Kusters, and M. Kist. 2000. Regulation of ferritin-mediated cytoplasmic iron
storage by the ferric uptake regulator homolog (Fur) of Helicobacter pylori. J. Bacteriol. 182:5948-5953. [Free Full text in PMC].
9.Bereswill, S., F. Lichte, T. Vey, F. Fassbinder, and M. Kist. 1998. Cloning and characterization of the fur gene from Helicobacter pylori. FEMS Microbiol. Lett.
159:193-200. [PubMed] [Full Text].
10.Blaser, M. J. 1992. Helicobacter pylori: its role in disease. Clin. Infect. Dis. 15:386-391. [PubMed]
11.Bode, G., F. Mauch, and P. Malfertheiner. 1993. The coccoid forms of Helicobacter pylori. Criteria for their viability. Epidemiol. Infect. 111:483-490. [PubMed].
12.Censini, S., C. Lange, Z. Xiang, J. E. Crabtree, P. Ghiara, M. Borodovsky, R. Rappuoli, and A. Covacci. 1996. cag, a pathogenicity island of Helicobacter pylori,
encodes type I-specific and disease-associated virulence factors. Proc. Natl. Acad. Sci. USA 93:14648-14653. [Free Full text in PMC].
13.Coghlan, J. G., D. Gilligan, H. Humphries, D. McKenna, C. Dooley, E. Sweeney, C. Keane, and C. O'Morain. 1987. Campylobacter pylori and recurrence of duodenal
ulcers—a 12-month follow-up study. Lancet 2:1109-1111. [PubMed].
14.Cover, T. L., C. P. Dooley, and M. J. Blaser. 1990. Characterization of and human serologic response to proteins in Helicobacter pylori broth culture supernatants
with vacuolizing cytotoxin activity. Infect. Immun. 58:603-610. [Free Full text in PMC].
15.Crabtree, J. E., R. L. Ferrero, and J. G. Kusters. 2002. The mouse colonizing Helicobacter pylori strain SS1 may lack a functional cag pathogenicity island.
Helicobacter 7:139-140. [PubMed] [Full Text].
16.de Boer, W. A. 1999. Bismuth triple therapy: still a very important drug regimen for curing Helicobacter pylori infection. Eur. J. Gastroenterol. Hepatol. 11:697-700.
[PubMed].
17.de Boer, W. A., and G. N. Tytgat. 2000. Regular review: treatment of Helicobacter pylori infection. Br. Med. J. 320:31-34. [PubMed] [Free Full Text].
18.Dhaenens, L., F. Szczebara, and M. O. Husson. 1997. Identification, characterization, and immunogenicity of the lactoferrin-binding protein from Helicobacter
pylori. Infect. Immun. 65:514-518. [Free Full text in PMC].
19.Domenico, P., L. Baldassarri, P. E. Schoch, K. Kaehler, M. Sasatsu, and B. A. Cunha. 2001. Activities of bismuth thiols against staphylococci and staphylococcal
biofilms. Antimicrob. Agents Chemother. 45:1417-1421. [Free Full text in PMC]
20.Domenico, P., D. R. Landolphi, and B. A. Cunha. 1991. Reduction of capsular polysaccharide and potentiation of aminoglycoside inhibition in gram-negative bacteria
by bismuth subsalicylate. J. Antimicrob. Chemother. 28:801-810. [PubMed].
21.Domenico, P., J. Reich, W. Madonia, and B. A. Cunha. 1996. Resistance to bismuth among gram-negative bacteria is dependent upon iron and its uptake. J.
Antimicrob. Chemother. 38:1031-1040. [PubMed].
22.Domenico, P., J. M. Tomas, S. Merino, X. Rubires, and B. A. Cunha. 1999. Surface antigen exposure by bismuth dimercaprol suppression of Klebsiella pneumoniae
capsular polysaccharide. Infect. Immun. 67:664-669. [Free Full text in PMC].
23.Emery, T. 1986. Exchange of iron by gallium in siderophores. Biochemistry 25:4629-4633. [PubMed].
24.Fekete, F. A., and L. L. Barton. 1991. Effects of iron(III) analogs on growth and pseudobactin synthesis in a chromiumtolerant Pseudomonas isolate. Biol. Met.
4:211-216. [PubMed].
25.George, P. M., C. Conaghan, H. B. Angus, T. A. Walmsley, and B. A. Chapman. 1996. Comparison of histological and biochemical hepatic iron indexes in the
diagnosis of genetic haemochromatosis. J. Clin. Pathol. 49:159-163. [PubMed].
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
26.Goodwin, C. S., P. Blake, and E. D. Blincow. 1986. The minimum inhibitory and bactericidal concentrations of antibiotics and anti-ulcer agents against Campylobacter pylordis. J. Antimicrob.
Chemother. 17:309-314. [PubMed].
27.Goodwin, C. S., B. J. Marshall, E. D. Blincow, D. H. Wilson, S. Blackbourn, and M. Phillips. 1988. Prevention of nitroimidazole resistance in Campylobacter pylori by coadministration of colloidal
bismuth subcitrate: clinical and in vitro studies. J. Clin. Pathol. 41:207-210. [PubMed].
28.Huang, C. T., and P. S. Stewart. 1999. Reduction of polysaccharide production in Pseudomonas aeruginosa biofilms by bismuth dimercaprol (BisBAL) treatment. J. Antimicrob. Chemother. 44:601605. [PubMed] [Free Full Text].
29.Keenan, J. I., and R. A. Allardyce. 2000. Iron influences the expression of Helicobacter pylori outer membrane vesicle-associated virulence factors. Eur. J. Gastroenterol. Hepatol. 12:1267-1273.
[PubMed].
30.Keenan, J. I., R. A. Allardyce, and P. F. Bagshaw. 1997. Dual silver staining to characterise Helicobacter spp. outer membrane components. J. Immunol. Methods 209:17-24. [PubMed] [Full Text].
31.Koo, J., J. Ho, S. K. Lam, J. Wong, and G. B. Ong. 1982. Selective coating of gastric ulcer by tripotassium dicitrato bism uthate in the rat. Gastroenterology 82:864-870. [PubMed].
32.Lambert, J. R., and P. Midolo. 1997. The actions of bismuth in the treatment of Helicobacter pylori infection. Aliment. Pharmacol. Ther. 11(Suppl. 1):27-33. [PubMed].
33.Lee, A., J. O'Rourke, M. C. De Ungria, B. Robertson, G. Daskalopoulos, and M. F. Dixon. 1997. A standardized mouse model of Helicobacter pylori infection: introducing the Sydney strain.
Gastroenterology 112:1386-1397. [PubMed] [Full Text].
34.Lee, F. I., I. M. Samloff, and M. Hardman. 1985. Comparison of tri-potassium di-citrato bismuthate tablets with ranitidine in healing and relapse of duodenal ulcers. Lancet 1:1299-1302. [PubMed].
35.Leunk, R. D., P. T. Johnson, B. C. David, W. G. Kraft, and D. R. Morgan. 1988. Cytotoxic activity in broth-culture filtrates of Campylobacter pylori. J. Med. Microbiol. 26:93-99. [PubMed].
36.Markwell, M. A., S. M. Haas, L. L. Bieber, and N. E. Tolbert. 1978. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem.
87:206-210. [PubMed].
37.Marshall, B. J., J. A. Armstrong, G. J. Francis, N. T. Nokes, and S. H. Wee. 1987. Antibacterial action of bismuth in relation to Campylobacter pyloridis colonization and gastritis. Digestion 37:1630. [PubMed].
38.McGowan, C. C., T. L. Cover, and M. J. Blaser. 1997. Analysis of F1F0-ATPase from Helicobacter pylori. Infect. Immun. 65:2640-2647. [Free Full text in PMC].
39.McNulty, C. A., J. Dent, and R. Wise. 1985. Susceptibility of clinical isolates of Campylobacter pyloridis to 11 antimicrobial agents. Antimicrob. Agents Chemother. 28:837-838. [Free Full text in
PMC].
40.Midolo, P. D., J. R. Lambert, T. G. Kerr, and W. Tee. 1999. In vitro synergy between ranitidine bismuth citrate and tetracycline or clarithromycin against resistant strains of Helicobacter pylori. Eur.
J. Clin. Microbiol. Infect. Dis. 18:832-834. [PubMed] [Full Text].
41.Odenbreit, S., J. Puls, B. Sedlmaier, E. Gerland, W. Fischer, and R. Haas. 2000. Translocation of Helicobacter pylori CagA into gastric epithelial cells by type IV secretion. Science 287:1497-1500.
[PubMed] [Full Text].
42.Ratledge, C., and L. G. Dover. 2000. Iron metabolism in pathogenic bacteria. Annu. Rev. Microbiol. 54:881-941. [PubMed] [Full Text]
.43.Rauws, E. A., W. Langenberg, H. J. Houthoff, H. C. Zanen, and G. N. Tytgat. 1988. Campylobacter pyloridis-associated chronic active antral gastritis. A prospective study of its prevalence and the
effects of antibacterial and antiulcer treatment. Gastroenterology 94:33-40. [PubMed].
44.Sox, T. E., and C. A. Olson. 1989. Binding and killing of bacteria by bismuth subsalicylate. Antimicrob. Agents Chemother. 33:2075-2082. [Free Full text in PMC].
45.Stoltenberg, M., M. Martiny, K. Sorensen, J. Rungby, and K. A. Krogfelt. 2001. Histochemical tracing of bismuth in Helicobacter pylori after in vitro exposure to bismuth citrate. Scand. J.
Gastroenterol. 36:144-148. [PubMed]
46.Stratton, C. W. 1996. Mechanisms of action for antimicrobial agents: general principles and mechanisms for selected classes of antibiotics, p. 579-603. In V. Lorian (ed.), Antibiotics in laboratory
medicine. Lippincott, Williams & Wilkins Co., Baltimore, Md.
47.Stratton, C. W., R. R. Warner, P. E. Coudron, and N. A. Lilly. 1999. Bismuth-mediated disruption of the glycocalyx-cell wall of Helicobacter pylori: ultrastructural evidence for a mechanism of
action for bismuth salts. J. Antimicrob. Chemother. 43:659-666. [PubMed] [Free Full Text].
48.Stryer, L. 1988. Biochemistry, 3rd ed. W. H. Freeman & Co., New York, N.Y
49.Tytgat, G. N. J., A. T. R. Axon, and M. F. Dixon. 1990. Helicobacter pylori: causal agent in peptic ulcer? Working Party Reports of the World Congress of Gastroenterology. Blackwell Scientific
Publications Ltd., Oxford, England.
50.Vaara, M., and T. Vaara. 1983. Polycations sensitize enteric bacteria to antibiotics. Antimicrob. Agents Chemother. 24:107-113. [Free Full text in PMC].
51.Van Caekenberghe, D. L., and J. Breyssens. 1987. In vitro synergistic activity between bismuth subcitrate and various antimicrobial agents against Campylobacter pyloridis (C. pylori). Antimicrob.
Agents Chemother. 31:1429-1430. [Free Full text in PMC].
52.Worst, D. J., B. R. Otto, and J. de Graaff. 1995. Iron-repressible outer membrane proteins of Helicobacter pylori involved in heme uptake. Infect. Immun. 63:4161-4165. [Free Full text in PMC].
53.Yeo, H. J., S. N. Savvides, A. B. Herr, E. Lanka, and G. Waksman. 2000. Crystal structure of the hexameric traffic ATPase of the Helicobacter pylori type IV secretion system. Mol. Cell 6:1461-1472.
Another YCP Controversy:
Genotipificación of isolated clinical of Helicobacter pylori on the basis of associated genes
to virulence . Correlation between Chilean types and others cultures(e.i.: Ethiopian)
• Chilean Study: Genotipificación of isolated clinical of
Helicobacter pylori on the basis of associated genes to
virulence cagA, vacA and babA2. First isolation of a
positive stock babA2.
• Ethiopian study: Correlation of Prevalence between
Helicobacter pylori vacA and cagA Genotypes in Others
cultures.
• Genotipificación of isolated clinical of Helicobacter pylori on the
basis of associated genes to virulence cagA, vacA and babA2. First
isolation of a positive stock babA2.
• Apolinaria Garcia C1a, Ricardo Barra T1b, Thin Carolina Sch2,
Fernando Kawaguchi P3, Natalia Trabal Fç, Sonia Montenegro H4d
and Carlos González C1e 1 Laboratory of Bacterial Pathogenicity.
Department of Microbiology, Faculty of Biological Sciences. 2
Department of Pathology and 3Departamento of Internal Medicine,
Medicine Faculty. University of Conception. Conception, Chile. to
4Molecular Immunogenetics Laboratory, Ochsner Clinic
Foundation, New Orleans, The USA.
•
Genotypying of clinical isolates of Helicobacter pylori by cagA, vacA and babA2 virulence associated genes. First
isolation of a babA2 positive strain.
•
SUMMARY
•
Background. Gastroduodenal diseases associated to Helicobacter pylori species depends on host characteristics,
environmental conditions and bacterial virulence factors, like cagA, vacA y babA2 genes product. Moreover, peptic
ulcer disease is related with cagA+, vacAs1m1 strains while H. pylori cagA+, vacAs1 y babA2+ strains are
associated to metaplasia and gastric cancer. Nonetheless, gene babA2 is still not described in clinical isolates from
Chilean patients.
•
Aim. To investigate the presence of cagA, vacA (s and m) and babA2 genes in clinical isolates of H. pylori among
Chilean patients.
•
Methods. Sixty six strains isolated from 41 patients were genotyped by PCR, using s1a, s1b, s2, m1, m2, cagA and
babA2 primers described elsewhere.
•
Results. cagA gene was detected in 16 isolates (24.2%) while vacAs1a, vacAs1b, vacAs2, vacAm1, and vacAm2
were detected in 28 strains (42.4%), 14 strains (21.2%), 17 strains (25.8%), 21 strains (31.8%) and 29 strains
(43.9%), respectively.
•
One strain (1.5%) was babA2 positive, being the first strain with this genotype described in Chile. This strain
presented a genotype cagA+, vacAs1 y babA2+ which is related with metaplasia or gastric cancer. In addition, five
strains showed an ulcerogenic profile cagA+, vacAs1m1.
•
Conclusion. The results presented indicate the prevalence of vacAs1m1 genotype among clinical strains analyzed,
and the presence of low frequency of strains with babA2 + genotype.
• Introduction.
• The development of gastroduodenales pathologies
associated to Helicobacter pylori has been associated to
characteristics of the guest, the medio.ambiente and
factors of virulence of the bacterium, such as codified by
the genes cagA, vacA and babA2.
• The ulcerous peptic disease is associated to stocks cagA+
and vacAs1m1, whereas the gastric presence of
metaplasia and adenocarcinoma is related to stocks of H.
pylori cagA+, vacAs1 and babA2+.
• This last gene not yet has been described in Chilean
stocks.
•
Objective.
•
Genotipificar isolated stocks of H. pylori of patients with gastroduodenal pathology, on the basis of
the genes cagA, vacA (region s and m) and babA2.
•
Methods.
•
66 stocks of H. were analyzed pylori obtained of gastric biopsies of 41 patients with high digestive
pathology.
•
he genotipificación was made by means of PCR, using partidores for s1a, s1b, s2, m1, m2, cagA
and babA2, described in Literature.
•
Results. Sixteen stocks presented/displayed the gene cagA (24.2%), 28 vacAs1a (42.4%), 14
vacAs1b (21.2%), 17 vacAs2 (25.8%), 21 vacAm1 (31.8%), 29 vacAm2 (43.9%) and one stock
(1.5%) presented/displayed the gene babA2.
Five stocks (7.6%) presented/displayed a genetic profile associate with ulcerogénicas stocks and
the stock babA2+ (1.5%) presented/displayed a genotype associated to metaplasia and/or
adenocarcinoma gastric.
•
•
The infection by Helicobacter pylori establishes in the region antropìlorica of the
stomach, in most of the infected individuals, a chronic, asintomática inflammation,
long play, possibly because the immunological defensive mechanisms of the guest fail
in their elimination (1, 2).
•
The persistence of the infection in certain patients is in the development of severe
pathologies, that progress of chronic gastritis to peptic ulcer, atrófica gastritis,
linfoma of gastric MALT and adenocarcinoma (3, 4, 5).
•
In spite of the high prevalence of the infection by H. pylori, only one minority of
infected individuals develops a malignant severe pathology.
This can be due to the genetic diversity between individuals (6);
•
•
•
to environmental factors, like type of diet, age of the first infection (7), and to specific
factors of virulence of the bacterium (8,9).
The gene cagA, that codifies a inmunodominante antigen, does not appear in all the
stocks of H. pylori (10), but comprises of the pathogenicity small barren island
(cagPAI), that contains 31 genes (11).
•
Therefore, its molecular detection indicates the presence of the PAI in the chromosome of the
microorganism (9).
•
The stocks cag+ (stocks type I) are associated to greater virulence, when inducing visible gastric
damage, whereas the stocks cag- (stocks type II) are associated to smaller virulence and they
behave like bacteria at the table than more pathogenic (1).
•
•
The vacuolizante citotoxina VacA is secretada by around 50% of the stocks of H. pylori and causes
degeneration to vacuolar of the epithelial gastric cells and ulceración of the gastric mucosa (12).
•
The citotoxina is codified by the gene vacA, that can present/display genetic mosaic on the basis of
alélicas variations in the regions mediates (alelos m1 or m2 and subtypes) and of signal (alelos s1
or s2 and subtypes) of the gene (13,14).
•
Specifically, one has demonstrated that stocks vacA s1/m1 have a high cytotoxic activity in
comparison with stocks s1/m2 and that stocks s2/m2 would not have cytotoxic activity (13).
• Recently one has demonstrated that factors of bacterial adhesion
contribute to the pathogenicity of H. pylori (15-17).
• Thus, the adhesina, codified by the gene babA2, favors a persistent
union between the microorganism and the gastric epithelial cell by
union of the bacterial cell through its BabA2 protein with group
antigen Lewis B (LeB) present in the gastric mucosa (2).
• Therefore, positive stocks of H. pylori babA2 present/display greater
capacity of adhesion, however the negative stocks babA2 adhere
weakly (18).
• This adhesion has been associated with high levels of linfocitaria
infiltration, intestinal glandular atrophy, metaplasia and increase of
the epithelial proliferation, reporting a significant association with
duodenal ulcer and gastric cancer (19).
• According to Yu and col. (2002) (17), the gene babA2 could be a
useful molecular marker to identify patients with greater risk of
presenting/displaying associate severe pathologies to infections by H.
pylori.
• Alves Oliveira and col. (2003) (20) in a study in Brazil informed a
strong association between babA2 and the presence into peptic ulcer
or gastric carcinoma.
•
It is important to consider that the frequency of virulence
determinants and its association with gastrointestinales pathologies
vary considerably in different geographic regions..
• The presence of more than one of these genes of
virulence would be associated with greater severity of the
gastric injury.
• One has inquired that stocks cagA+, vacAs1m1 would be
associate of ulcers and stocks triple upon presentment
positive cagA+, vacAs1,
• babA2+ in addition to ulcer, would be associated with
metaplasia and adenocarcinoma gastric (21, 22).
• In Chile more of 90% of the patients with peptic ulcer
and the 42 % of those who consult by dispepsia
nonulcerosa they are infected with H. pylori (23).
• In spite of the high prevalence of infection by H. pylori in
our country, still the works in genotipificación of stocks
including the genes are few cagA and vacA (24; 25; 26)
and are not works with respect to the gene babA2.
• The objective of the present study was to genotipificar
isolated stocks of H. pylori of patients with
gastroduodenal pathology, on the basis of the genes
cagA, vacA (region s and m) including the gene babA2
not detected previously in Chilean stocks
• Conclusion.
• The results as a whole indicate the predominance
of stocks with genotype vacAs1m1 between
isolated clinical of H. pylori and a LF in the
detection of the positive genotype babA2.
• This gene could be explored as marking of more
severe gastric injuries in our means.
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
Referencias
TAYLOR D, BLASER M J. The epidemiology of Helicobacter pylori infection. Epidemiol Rev 1991; 13: 42-59.
NILSSON C, SILLEN A, ERIKSSON L, STRAND ML, ENROTH H, NORMARK S, FALK P, ENGSTRAND L. Correlation between cag pathogenicity island
composition and Helicobacter pylori-associated gastroduodenal diseases. Infec Immun 2003; 71: 6573-81.
MARSHALL B. Helicobacter pylori. Am J Gastroenterol. 1994; 89: 116-27.
FOREMAN D, AND THE EUROGAST STUDY GROUP, An International Association between Helicobacter pylori infection and gastric cancer. Lancet
1993; 341: 359-62.
WOTHERSPOON A, DOGLIONI C, DISS T, PAN L, MOSCHINI A, DE BONI M, ISAACSON P.. Regression of primary low-grade B-cell gastric lymphoma
of mucosa-associated lymphoid tissue type after eradication of Helicobacter pylori. Lancet.1993;342: 575-77.
AZUMA T, ITO S, SATO F, YAMAZAKI Y, MIYAJI H, ITO Y, SUTO H, KURIYAMA M, KATO T, KHOLI Y. The role of the HLA-DQA1 gene in resistance to
atrophic gastritis and gastric adenocarcinoma induced by Helicobacter pylori infection. Cancer 1998; 82: 1013-18.
KATO I, VIVAS J, PLUMMER M, LOPEZ G, PERAZA S, CASTRO D, SANCHEZ V, CANO E, ANDRADE O, GARCIA R, FRANCESCHI S, OLIVER W,
MUNOZ N. Environmental factors in Helicobacter pylori-related gastric precancerous lesions in Venezuela. Cancer Epidemiol Biomarkers Prev 2004; 13:
468-76.
YANG J, WANG T, WANG H, KUO C, WANG J, WANG W. Genetic analysis of the cytotoxin associated gene and the vacuolating gene in Helicobacter pylori
strains isolated from Taiwanese patients. Am J Gastroenterol 1997; 92: 1316-21.
BLASER MJ, ATHERTON JC. Helicobacter pylori persistence: biology and disease. J Clin Invest 2004; 113: 321-33.
COVACCI A, CENSINI S, BUGNOLI M. Molecular characterization of the 128-kDa immunodominant antigen of Helicobacter pylori associated with
cytotoxicity and duodenal ulcer. Proc Natl Acad Sci USA 1993; 90: 5791-95.
11. CENSINI S, LANGE C, XIANG Z, CRABTREE J, GHIARA P, BORODOVSKY M, RAPPUOLI R AND COVACCI A. Cag, a pathogenicity island of
Helicobacter pylori, encoded type I-specific and disease-associated virulence factors. Proc Natl Acad Sci USA. 1996 ; 93: 14648-53.
12. COVACCI A, TELFORD JL, DEL GIUDICE G, PARSONNET J, RAPPUOLI R. Helicobacter pylori virulence and genetic geography. Science 1999;
284: 1328-33.
13. ATHERTON JC, CAO P, PEEK RM JR, TUMMURU MK, BLASER MJ, COVER TL. Mosaicism in vacuolating cytotoxin alleles of Helicobacter pylori.
Association of specific vacA types with cytotoxin production and peptic ulceration. J Biol Chem 1995; 270: 1771-7.
14. VAN DOORN LJ, FIGUEIREDO C, SANNA R, PENA AS, MIDOLO P, ATHERTON JC, BLASER MJ, QUINT W. Expanding allelic diversity of
Helicobacter pylori vacA. J Clin Microbiol 1998; 36: 2597-2603.
15. MIZUSHIMA T, SUGIYAMA T, KOMATSU Y, ISHIZUKA J, KATO M, ASAKA M. Clinical relevance of the babA2 genotype of Helicobacter pylori in
Japanese clinical isolates. J Clin Microbiol 2001; 39: 2463-65.
16. LAICH, KUO CH, CHEN YC, CHAO FY, POON SK, CJANG CS, WANG WC. High prevalence of cagA- and babA2-positive Helicobacter pylori clinical
isolates in Taiwan. J Clin Microbiol 2002; 40:3860-62.
17. YU J, LEUNG WK, GO MYY, CJHAN MCW, TO KF, NG EKW, CHAN FKL, LING TKW, CHUNG SCS, SUNG JJY. Relationship between Helicobacter
pylori babA2 status with gastric epithelial cell turnover and premalignant gastric lesions. Gut 2002; 51: 480-84.
18. BOREN T, NORMARK S, FALK P. Helicobacter pylori: molecular basis for host recognition and bacterial adherence. Trends Microbiol 1994; 2: 22128.
19. GERHARD M, LEHN N, NEUMAYER N. ET AL Clinical relevance of the Helicobacter pylori gene for blood-group antigen-binding adhesin. Proc Natl
Acad Sci 1999; 96:1278-83.
20. ALVES O A, SANTOS A, BECATTINI G J, AGUIAR R G,
CAMARGOS R A, OLIVEIRA C, DEMAS A, CABRAL M, FERREIRA N A y MAGALAHES Q D. babA2- and cagA-Positive Helicobacter pylori Strains Are
Associated with Duodenal Ulcer and Gastric Carcinoma in Brazil. J Clin Microbiol 2003; 41: 3964–66.
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
21. GERHARD M, LEHN N, NEUMAYER N ET AL. Clinical relevance of the Helicobacter pylori gene for blood-group antigen-binding adhesin. Proc Natl Acad Sci USA.
1996; 96: 12778-83.
22. ZAMBON CF, NAVAGLIA F, BASSO D, RUGGE M, PLEBANI M. Helicobacter pylori babA2, cagA, and s1vacA genes work synergistically in causing intestinal
metaplasia. J Clin Pathol 2003; 56: 287-91.
23. PRADO, V. Enfermedades infecciosas emergentes: ¿Un problema nuevo? Rev Med Chile 1996; 124: 7-10.
24. MARTÍNEZ A, GONZÁLEZ C, KAWAGUCHI F, MONTOYA R, CORVALÁN A, MADARIAGA J, ROA J, GARCÍA A, SALGADO F, SOLAR H, PALMA M.
Helicobacter pylori: análisis de cagA y genotipificación de vacA en Chile. Detección de una cepa s2/m1. Rev Med Chile 2001; 129: 1147-53.
25. FAÚNDEZ G, TRONCOSO M, FIGUEROA G. cagA and vacA in strains of Helicobacter pylori from ulcer and non-ulcerative dyspepsia patients. BMC Gastroenterol
2002, 2: 20.
26. ARAYA J, ANABALÓN L, ROA I, BRAVO M, VILLASECA M, GUZMÁN P, ROA J. Relación de la genotipificación de Helicobacter pylori con la forma e intensidad
de la gastritis en población adulta portadora de patología gástrica benigna. Rev Med Chile 2004; 132: 1345-54.
27. WORKING PARTY OF THE EUROPEAN HELICOBACTER PYLORI STUDY GROUP. Guidelines for clinical trials in Helicobacter pylori infection. Gut 1997; 41
(Suppl 2): S3.
28. MAZURIER S, VAN DE GIESSEN A, HEUVELMAN K, WERNARS K. RAPD analysis of Campylobacter isolates: DNA fingerprinting without the need to purify DNA.
Lett Appl Microbiol 1992; 14: 260-62.
29. TUMMURU MK, COVER TL, BLASER MJ. Cloning and expression of a high-molecular-mass major antigen of Helicobacter pylori: evidence of linkage to cytotoxin
production. Infect Immun 1993; 61: 1799-809.
30. SALGADO F, GARCÍA A, OÑATE A, GONZÁLEZ C, KAWAGUCHI F. Increased in-vitro and in-vivo biological activity of lipopolysaccharide extracted from clinical
low virulence vacA genotype Helicobacter pylori strains. J Med Microbiol 2002; 51: 771-76.
31. NAVAGLIA F, BASSO B y PLEBANI M. Touchdown PCR: a rapid method to genotype Helicobacter pylori infection. Clin Chim Acta 1997; 262: 57-60.
32. PODZORSKI R, PODZORSKI D, WUERTH A, TOLIA V. Analysis of the vacA, cagA, cagE, iceA and babA2 genes in Helicobacter pylori from sixty-one pediatric
patient from the Midwestern united states. Diagnos Microbiol Infec Dis. 2003, 46: 83-88.
33. HEHENBERGER P, GRETECHEL S. Gastric Cancer. The Lancet, 2003. Tomo 362, N 9380: pp 305.
34. KIM S-Y, WOO C, LEE Y-M, SON B, KIM J, CHAE H, YOUN S, PARK S. Genotyping cagA, vacA subtype, iceA and babA of Helicobacter pylori isolates from Korean
patients, and their association with gastroduodenal diseases. J Korean Med Sci 2001; 16: 579-84.
35. LAI C-H, KUO C-H, CHEN Y-C, CHAO F-Y, POON S-K, CHANG C-S AND WANG W-C. High prevalence of cagA and babA2 positive Helicobacter pylori clinical
isolates in Taiwan. J Chin Med Assoc 2002; 40: 3860-62.
36. YAKOOB J, FAN X G, PENG X N, HU G L, ZHANG Z. Helicobacter pylori cagA and vacA cytotoxin genes in Changsha, China. Br J Biomed Sci 2002; 59: 150-3.
37. YU J, LEUNG WK, GO MYY, CHAN MCW, TO KF, NG EKW, CHAN FKL, LING TKW, CHUNG SCS, SUNG JJK. Relationship between Helicobacter pylori babA2
status with gastric epithelial cell turnover and premalignant gastric lesions. Gut. 2002; 51: 480-484.
Correlation of Prevalence between Helicobacter pylori vacA and cagA
Genotypes in Others cultures (e.i. : Ethiopian)
DRA. ALEJANDRA MARTINEZ, IGNACIO ALFARO, TAMARA PEREZ, CINTHYA PEREZ.
• A total of 300 gastric biopsy samples and 50 Helicobacter pylori isolates were
collected from Ethiopian adult dyspeptic patients.
• The vacA and cagA genes were detected in 90 and 79% of biopsy specimens,
• respectively, and in 100 and 87% of clinical isolates, respectively. Both genes
were detected in 84% of the gastric biopsy samples and in 87% of the clinical
isolates.
• Among vacA genotypes, the s1/m1 genotype was the most common in gastric
biopsy samples (48%).
• The vacA and cagA positive H. pylori strains were
• detected to a higher degree in patients with chronic active gastritis (71%) than
patients with other histopathological findings (29%) (P < 0.05).
•
•
•
•
•
•
•
Several Helicobacter pylori virulence genes related to the risk
of gastroduodenal diseases have been proposed.
The vacuolating
cytotoxin (vacA) gene is present in virtually all H. pylori
strains and contains at least two variable regions, the signal (s)
region, which encodes the signal peptide, and the middle (m)
region (4).
•
•
•
The s region has been divided into two subtypes, s1
and s2, and the m region has been divided into two subtypes,
m1 and m2 (19).
•
•
•
•
•
•
•
•
The amount of cytotoxin produced is highest
with the s1/m1 allele, followed by the s1/m2 allele, while no
cytotoxin activity is found when s2/m2 is present (19).
The
cytotoxin-associated gene (cagA) is a marker for a genomic
pathogenicity island of 40 kb (6). A significant association
between the presence of ulcers or gastric carcinoma and the
presence of vacA type s1 and cagA gene (5, 19).
•
•
•
•
The present
study represents the first in Ethiopia to detect H. pylori vacA
and cagA genotypes from gastric biopsy samples and clinical
isolates using PCR-based methods.
MATERIALS AND METHODS
•
•
•
•
•
•
•
•
•
•
•
•
Study subjects. A total of 300 consecutive informed and consenting adult
patients with dyspeptic symptoms from the gastrointestinal referral and followup clinics of Department of Internal Medicine, Tikur Anbassa University
Hospital, Addis Ababa, Ethiopia, were investigated for H. pylori between November
2000 and August 2002. The mean age of the patients was 36.5 years
(standard deviation, 13.8 years; range, 15 to 90 years). The majority of patients
(76%) were between the ages of 15 and 44 years. Of the 300 patients, 186 (62%)
were males and 114 (38%) were females (resulting in an overall male to female
ratio of 1.6:1).
The study was approved by the Department Graduate Committee, the Faculty
Research Publications Committee and endorsed by the Faculty Academic Commission
and has been ethically cleared.
Culture and identification
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
. Antral gastric biopsy samples were taken from
each dyspeptic patient. The biopsy specimens were put into sterile phosphatebuffered
saline containing 15% glycerol and immediately transported to laboratory
for culture. Biopsy samples for molecular analysis were kept frozen in 15%
tryptone soy broth (Oxoid Ltd., Basingstoke, England) and stored at _70°C until
analyzed.
H. pylori was cultured from antral biopsy specimens using a standard method
(17). H. pylori identification was based on morphology, Gram staining, oxidase,
catalase, and urease tests.
All the isolated H. pylori strains were kept frozen at
_70°C in the tryptone soy broth medium containing 15% (vol/vol) glycerol until
genotyping was performed.
The H. pylori reference strain (CCUG 17874) (Culture
Collection, University of Gothenburg, Gothenburg, Sweden) was cultured
throughout the study for quality control.
Histopathology. Gastric biopsy specimens were fixed in 10% formalin and
embedded in paraffin. The sections (4 to 5 _m thick) were cut and stained with
hematoxylin and eosin (2). The histological findings from the sections stained
with hematoxylin and eosin were scored according to the updated Sydney system
of classification and grading of gastritis (7).
Genomic DNA extraction. Biopsy specimens and isolates were centrifuged at
•
•
•
•
•
•
•
•
10,000 _ g for 5 min. The DNA was extracted from the pellets by use of the
QIAamp DNA kit (QIAGEN, Hilden, Germany) according to the manufacturer’s
recommendations and DNA stored at _20°C until analysis. DNA extraction
negative controls were performed in parallel by including sterile tubes without
samples to check for contamination of the DNA extraction reagents.
PCR-DGGE.
The PCR amplification was carried out using a GeneAmp 2700
Thermal cycler (Applied Biosystems, Foster City, Calif.).
•
•
•
•
•
•
A seminested Helicobacter genus-specific PCR assay targeting the 16S rDNA was used to amplify
Helicobacter DNA (9).
Denaturing gradient gel electrophoresis (DGGE) analysis
of the PCR products was performed in a DCode system (Bio-Rad, Hercules,
Calif.) as recently described (1).
Migration ladder containing PCR products of reference Helicobacter strains (H. muridarum [CCUG 29262], H.
bilis [CCUG
38995], H. pullorum [NCTC 12825], H. pylori [CCUG 17874], “Flexispira rappini”
[CCUG 23435], H. hepaticus [CCUG 33637], and H. bizzozeronii [AF 53]) was
run in parallel as a mobility ladder.
vacA and cagA genotyping.
•
•
•
•
•
•
•
•
Detection of H. pylori vacA and cagA genes was performed on gastric biopsy specimens and isolates positive for H.
pylori by PCR-DGGE as previously described (15, 19).
As a positive control, H. Pylori (CCUG 17874) DNA (_0.1 ng) was added to the reaction mixture, while 5 _l of
sterile deionized Millipore-filtered water was added to the reaction mixture as a negative control. Estimation of size
of the PCR products was done by using Gene ruler 100-bp DNA ladder (Fermentas, Vilnius, Lithuania). The
products of each PCR assay were visualized by electrophoresis in a 1.5% agarose gel containing
ethidium bromide (15).
RESULTS AND DISCUSSION
•
•
•
Histopathological examinations were performed on 276
(92%) of the gastric biopsy specimens, whereas the remaining
24 (8%) were not adequate (too small).
•
Abnormal findings were observed in all examined specimens.
•
•
Chronic gastritis was found in 48 (17.4%) patients, chronic active gastritis in 185
(67%), chronic atrophic gastritis in 24 (9%);
•
•
chronic atrophic gastritis with intestinal metaplasia in 17 (6%) and malignant
lesions in 2 (0.6%) patients.
•
•
The overall prevalence of chronic gastritis was 99.3%. The most common histopathological
findings in the present study were similar to those reported from
other parts of Africa (10).
•
•
•
figures reported from The Netherlands (36%), Hong Kong (26
to 31%), and Nigeria (24%), but lower than figures reported
from Brazil (80%) and Korea (78%) (3, 11, 16, 20, 21). In the
• The seminested Helicobacter genus-specific PCR assay detected Helicobacter DNA
in 273 of 300 (91%) of the biopsy specimens. DGGE analysis showed that all PCR
products have
• mobility pattern similar to the H. pylori reference strain (Fig. 1).
• The vacA gene was detected in 246 of 273 (90%) of H. pylori-positive gastric biopsy
specimens, which is similar to reported results from The Netherlands (93%) and
Hong Kong
• (95.8%) (14, 21), emphasizing high sensitivity of the PCR method employed in the
present study.
• The vacA genotypes s1/m1, s1/m2, s2/m2, and s2/m1 were found in 48, 28, 9, and
2% of the specimens, respectively (Table 1), whereas, 15 biopsy specimens (6%)
were incomplete and thus did not yield a detectable PCR product for the vacA s or m
regions.
• The pattern of vacA alleles in this study is in agreement with those reported in other
studies (3, 8, 16, 18, 20).
• However, the frequencyof vacA s1/m1 allelic type in this study is higher than
•
•
•
In the present investigation, the rare vacA s2/m1 allele was detected
in 4 (2%) of the 246 gastric biopsy specimens examined, also
reported in studies in South Africa and Chile (12, 13).
•
•
•
Multiple vacA genotypes were found in 18 (7%) of the 246-biopsy specimens
examined. The most frequent multiple vacA genotypes were s1/m1m2 (11 of 18; 61%).
The vacA was detected in all 52 H. pylori isolates tested (Table 1).
•
The prevalence of the vacA subtypes s1/m1, s1/m2, and s2/m2 was 60, 27, and 7%,
respectively.
Three (6%) of the isolates contained mixed vacA subtypes; two s1/m1m2 and one
s1s2/m2 from a single H. pylori isolate.
The multiple vacA genotypes detected in this study are similar to results from Italy
reported by Blaser and Berg (5).
Surprisingly, the prevalence of multiple vacA genotypes in this study was much lower
compared with results reported from Brazil (13%), Chile (32%), Korea (18%), and The
Netherlands (11%) (3, 11, 13, 20).
•
•
•
•
•
•
The low prevalence of multiple vacA genotypes in a country with a high prevalence of H.
pylori infections in the general population may be the result of a low number of mosaics
of any combination of signal (s) and mid-region (m) alleles of the bacteria circulating in
the community.
• Of the 273 H. pylori PCR-positive biopsy specimens, 217
• (79%) were cagA positive.
• Four different genotypic combinations were recognized based on analysis of
the positive and negative vacA and cagA results—vacA_ cagA_, vacA_ cagA,
vacA cagA, and vacA cagA_, which were found in 76, 14, 6, and 4% of
specimens, respectively (Table 2).
• Forty-five of the 52 (87%) H. pylori strains were positive for both vacA and
cagA, whereas the remaining isolates 7 (13%) were only vacA positive(Table 2).
• Statistical analysis showed no difference in the detection of the vacA and cagA
in gastric biopsy specimens and clinical isolates (P 0.70 for vacA, P 0.96 for
cagA).
• In
• addition, no statistical differences in the frequency of detection
• of the different vacA allelic types from gastric biopsy specimens
• and clinical isolates were found (P _ 0.05).
• The prevalence of
• The prevalence of cagA positive H. pylori strains varies from one geographic region
• to another, e.g., 38% in Chile, 48% in Sri Lanka, 67% in The Netherlands, 81% in
the United Sates, 90% in Hong Kong, 97% in Korea, 93% in Nigeria and 94% in
Brazil (3, 8, 11, 13, 16, 18, 20, 21).
• Correlation of histopathology results with vacA and cagA genotypes showed that
vacA and cagA positive strains were detected to a higher degree in patients with
chronic active gastritis (71%) compared with patients with
• other histopathological findings (29%) (P _ 0.05) (Table 3).
•
•
•
•
Molecular analyses demonstrated that more than 80% of the
FIG. 1. DGGE analysis of PCR products amplified by using Helicobacter
genus-specific primers.
Lanes, 1 to 4 and 6 to 22, H. Pyloripositive samples; 5, H. pylori-negative sample;
M, mobility marker
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
REFERENCES
1. Abu Al-Soud, W., M. Bennedsen, S. L. W. On, I.-S. Ouis, P. Vandamme,
H.-O. Nilsson, A. Ljungh, and T. Wadstro¨m. 2003. Assessment of PCRDGGE
for the identification of diverse Helicobacter species, and application
to faecal samples from zoo animals to determine helicobacter prevalence.
J. Med. Microbiol. 52:765–771.
2. Anim, J. T., N. Al-Sobkie, A. Prasad, B. John, P. N. Sharma, and I. AlHamar. 2000. Assessment of different methods for staining Helicobacter
pylori in endoscopic gastric biopsies. Acta Histochem. 102:129–137.
3. Ashour, A. A., P. P. Magalhaes, E. N. Mendes, G. B. Collares, V. R. de
Gusmao, D. M. Queiroz, A. M. Nogueira, G. A. Rocha, and C. A. de Oliveira.
2002. Distribution of vacA genotypes in Helicobacter pylori strains isolated
from Brazilian adult patients with gastritis, duodenal ulcer or gastric carcinoma.
FEMS Immunol. Med. Microbiol. 33:173–178.
4. Atherton, J. C., P. Cao, R. M. Peek, Jr., M. K. R. Tummuru, M. J. Blaser,
and T. L. Cover. 1995. Mosaicism in vacuolating cytotoxin alleles of Helicobacter
pylori. J. Biol. Chem. 270:17771–17777.
5. Blaser, M. J., and D. E. Berg. 2001. Helicobacter pylori genetic diversity and
risk of human disease. J. Clin. Investig. 107:767–773.
6. Censini, S., C. Lange, Z. Xiang, J. E. Crabtree, P. Ghiara, M. Borodovsky,
R. Rappuoli, and A. Covacci. 1996. Cag, a pathogenicity island of Helicobacter
pylori, encodes type I-specific and disease-associated virulence factors.
Proc. Natl. Acad. Sci. USA 93:14648–14653.
7. Dixon, M. F., R. M. Genta, J. H. Yardley, and P. Correa. 1996. Classification
and grading of gastritis. The updated Sydney system. International Workshop
on the Histopathology of Gastritis, Houston 1994. Am. J. Surg Pathol.
20:1161–1181.
8. Fernando, N., J. Holton, D. Vaira, M. DeSilva, and D. Fernando. 2002.
Prevalence of Helicobacter pylori in Sri Lanka as determined by PCR. J. Clin.
Microbiol. 40:2675–2676.
9. Goto, K., H. Ohashi, A. Takakura, and T. Itoh. 2000. Current status of
helicobacter contamination of laboratory mice, rats, gerbils, and house musk
shrews in Japan. Curr. Microbiol. 41:161–166.
10. Kidd, M., J. A. Louw, and I. N. Marks. 1999. Helicobacter pylori in Africa:
observations on an
enigma within an enigma’. J. Gastroenterol. Hepatol.
14:851–858.
11. Kim, S. Y., C. W. Woo, Y. M. Lee, B. R. Son, J. W. Kim, H. B. Chae, S. J.
Youn, and S. M. Park. 2001. Genotyping cagA, vacA subtype, iceA1, and
babA of Helicobacter pylori isolates from Korean patients, and their association
with gastroduodenal diseases. J. Korean Med Sci. 16:579–584.
12. Letley, D. P., A. Lastovica, J. A. Louw, C. J. Hawkey, and J. C. Atherton.
1999. Allelic diversity of the Helicobacter pylori vacuolating cytotoxin gene in
South Africa: rarity of the vacA s1a genotype and natural occurrence of an
s2/m1 allele. J. Clin. Microbiol. 37:1203–1205.
•
•
•
•
13. Martinez, A., C. Gonzalez, F. Kawaguchi, R. Montoya, A. Corvalan, J.
Madariaga, J. Roa, A. Garcia, F. Salgado, H. Solar, and M. Palma. 2001.
Helicobacter pylori: cagA analysis and vacA genotyping in Chile. Detection
of a s2/m1 strain. Rev. Med. Chil. 129:1147–1153.
•
•
•
•
14. Scholte, G. H., L. J. van Doorn, W. G. Quint, and J. Linderman. 2001.
Genotyping of Helicobacter pylori strains in formalin-fixed or formaldehydesublimatefixed paraffin-embedded gastric biopsy specimens. Diagn. Mol.
Pathol. 10:166–170.
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•
15. Sjunnesson, H., T. Falt, E. Sturegard, W. Abu Al-Soud, A. Ljungh, and T.
Wadstro¨m. 2003. PCR-denaturing gradient gel electrophoresis and two feces
antigen tests for detection of Helicobacter pylori in mice. Curr. Microbiol.
47:278–285.
16. Smith, S. I., C. Kirsch, K. S. Oyedeji, A. O. Arigbabu, A. O. Coker, E.
Bayerdoffer, and S. Miehlke. 2002. Prevalence of Helicobacter pylori vacA,
cagA and iceA genotypes in Nigerian patients with duodenal ulcer disease.
J. Med. Microbiol. 51:851–854.
17. Soltesz, V., B. Zeeberg, and T. Wadstro¨m. 1992. Optimal survival of Helicobacter
pylori under various transport conditions. J. Clin. Microbiol. 30:1453–
1456.
18. Straus, E. W., H. Patel, J. Chang, R. M. Gupta, V. Sottile, J. Scirica, G.
Tarabay, S. Iyer, S. Samuel, and R. D. Raffaniello. 2002. H. pylori infection
and genotyping in patients undergoing upper endoscopy at inner city hospitals.
Dig. Dis. Sci. 47:1575–1581.
19. van Doorn, L. J., C. Figueiredo, R. Rossau, G. Jannes, M. van Asbroeck, J. C.
Sousa, F. Carneiro, and W. G. V. Quint. 1998. Typing of Helicobacter pylori
vacA gene and detection of cagA gene by PCR and reverse hybridization.
J. Clin. Microbiol. 36:1271–1276.
20. van Doorn, L. J., C. Figueiredo, R. Sanna, A. Plaisier, P. Schneeberger, W.
de Boer, and W. Quint. 1998. Clinical relevance of the cagA, vacA, and iceA
status of Helicobacter pylori. Gastroenterology 115:58–66.
21. Wong, B. C., Y. Yin, D. E. Berg, H. H. Xia, J. Z. Zhang, W. H. Wang, W. M.
Wong, X. R. Huang, V. S. Tang, and S. K. Lam. 2001. Distribution of distinct
vacA, cagA and iceA alleles in Helicobacter pylori in Hong Kong. Helicobacter
6:317–324.
Departamento de Microbiología
Facultad de Ciencias Biológicas
Universidad de Concepción
CITOMETRÍA DE FLUJO EN LA EVALUACIÓN DE
POTENCIAL DE MEMBRANA Y VIABILIDAD CELULAR
DE Helicobacter pylori.
T.M. Juan Luis Castillo Navarrete,
Dra Apolinaria García