Evaluation of culture media for Paenibacillus larvae applied to studies of antimicrobial activity
L. B. Gende*1, 2, 3, M. J. Eguaras1, 3, R. Fritz2
1Departamento de Biología y
2Departamento de Química, Facultad de Ciencias Exactas y Naturales.
3CONICET. Universidad Nacional de Mar del Plata.
Funes 3350 (7600) Mar del Plata, Pcia. de Buenos Aires, Argentina.
*Correspondencia. E-mail: lgende@mdp.edu.ar
ABSTRACT
This study was conducted to compare different liquid culture media for Paenibacillus larvae growth in order to find the best one to be used in studies on activity of antimicrobial substances, such as essential oils. P. larvae presented poor growth in usual broths such as Mueller-Hinton, commonly employed in antimicrobial activity assays. Growth in liquid media was evaluated using Paenibacillus larvae strains isolated from hives located in different geographical zones. The MYT medium (Mueller-Hinton broth, yeast extract and thiamine) was selected out of the eight liquid media analyzed, as it proved to be the most adequate due to its higher absorbance at 620 nm. The following mean values were obtained from the four P. larvae strains: 0.227 ± 0.016 for the Cobo strain, 0.279 ± 0.015 for La Plata strain, 0.758 ± 0.020 for Mechongué strain and 0.244 ± 0.0079 for Sierra de los Padres strain, respectively.
Key words: Antimicrobial activity; Culture media; Paenibacillus larvae
RESUMEN
Evaluación de medios de cultivo para el crecimiento de Paenibacillus larvae aplicables en estudios de actividad antimicrobiana. Este trabajo está orientado a comparar diferentes medios de cultivo líquidos para el crecimiento de Paenibacillus larvae. El objetivo fue encontrar el más apropiado para utilizar en estudios de actividad antimicrobiana de diferentes sustancias, tales como aceites esenciales. P. larvae presenta un crecimiento débil en medios de cultivo como el Mueller-Hinton, comúnmente usado en ensayos de actividad antimicrobiana. Se evaluó el crecimiento en caldos de cultivo de cepas aisladas de colmenas ubicadas en diferentes zonas geográficas. De los ocho medios analizados, el MYT (Mueller-Hinton, extracto de levadura y tiamina) mostró ser el más apropiado, en éste se observó el mayor valor de absorbancia a 620 nm. Los valores obtenidos en promedio para los cuatro aislamientos de P. larvae evaluados fueron 0,227 ± 0,016 (cepa de Cobo); 0,279 ± 0,015 (cepa de La Plata); 0,758 ± 0,020 (cepa de Mechongué) y 0,244 ± 0,0079 (cepa de Sierra de los Padres).
Palabras clave: Actividad antimicrobiana; Medios de cultivo; Paenibacillus larvae
American Foulbrood (AFB) is one of the most widespread
and destructive brood diseases which affects the
honey bee larval stage ( Apis mellifera). The causative
agent is Paenibacillus larvae (7), a gram-positive and
spore forming bacterium that infects queen, drone, and
worker larvae alike. Adult bees, however, are not affected
by AFB.
The growth of this bacterium occurs in two forms: vegetative
(rod-shaped bacterial cells) and spores. Only the
spore stage is infectious to honey bees. The spores germinate
into the vegetative stage soon after they enter the larval
gut, and continue to multiply until larval death. AFB is
one of the few bee diseases capable of killing a colony. The
prevention and control of this disease have features of its
own, as the spores can remain viable for long periods and
survive under adverse environmental conditions (10, 11).
While P. larvae sporulates and multiplies efficiently in
the hemolymph of bee larvae, most strains grow poorly in
or on artificial media. Different culture media have been
developed based on larval debris, honey, wax and adult
bees in order to isolate P. larvae. Several of them were
especially evaluated with the purpose of analyzing the
spore recovery of this species from honey, not only for
examining their composition but also the antibiotic addition,
and the assessment of different incubation conditions.
The MYPGP developed by Dingman and Stahly
(4) and made up of yeast extract, Mueller-Hinton broth,
glucose, K2HPO4, sodium pyruvate and agar, yielded the
highest percentage of spore recovery; while J agar, brain
heart agar fortified with thiamine, Columbia agar supplemented
with ovine blood, and agar supplemented with
horse blood proved to be less efficient in this respect (12).
Continued researches have also been underway to
optimize culture conditions so as to obtain maximum P.
larvae recovery from honey. To this end, different concentrations
of nalidixic and pipemidic acids antibiotics were tested. The conclusion drawn was that MYPGPNALPIA
B (9 µg/ml of nalidixic acid and 20 µg/ml of pipemidic acid)
is the most appropriate medium for honey spore recovery
within heterogeneous populations. Conversely, and
in connection with vegetative cell cultures, the most appropriate
media would be MYPGP and MYPGPNALPIA (6 µg/ml of nalidixic acid and 10 µg/ml of pipemidic acid)
(13). The AFB spore isolation from adult bees (9) employed
a J medium with 3 µg/ml of nalidixic acid, and (5)
used MYPGP agar supplemented with the same amount
of antibiotic.
Yet, further research should be conducted on liquid
media so as to favor the evaluation of vegetative cell
growth of this microorganism by turbidimetry (4, 8), and,
at the same time, the analysis of antimicrobial activity by
serial dilution. There are no recommendations available
by NCCLS regarding P. larvae CIM determination; and,
particularly, this bacterium finds it difficult to grow in
Mueller-Hinton liquid broth (6), the usually employed
medium in assays of this sort. In earlier studies on the
antimicrobial activity of antimicrobial substances, such as
essential oils, in which serial dilution methods with BHIT,
Mueller-Hinton broth, and Mueller-Hinton broth with thiamine
were employed, several problems were encountered.
The bacterial growth obtained was non-reproducible
among isolations for the first medium and poor for
the last two ones, which hindered the observation of the
inhibition resulting from the antimicrobial substances, and,
hence, the minimal inhibitory concentration measure.
The aim of this work was to obtain a suitable medium
applicable to studies of antimicrobial substances, such
as essential oils, against P. larvae.
Bacterial strains of P. larvae were isolated from honeycombs
exhibiting clinical symptoms of American
Foulbrood. These hives were located in the outskirts of
the cities of Mechongué, La Plata, Cobo and Sierra de
los Padres, all of them located within Buenos Aires province.
Isolation was achieved on MYPGP agar supplemented
with 9 µg/ml of nalidixic acid in order to inhibit the
growth of Paenibacillus alvei. Plates were incubated under
microaerobic conditions (5-10% of CO2), and the
strains were identified by employing standard biochemical
tests (2). The different strains of P. larvae were stored
at -80 °C on liquid MYPGP with 15% v/v of glycerol until
Vegetative cells of P. larvae were grown on MYPGP
agar incubating 48 h at 36±0.5 °C under aerobic conditions.
Afterwards, they were suspended in double distilled
sterile water and standardized to a turbidity level
approximating 107-108 cells/ml (absorbance of 0.258 at a
wave length of 620 nm) measured with a spectrophotometer
&Lomb Spectronic 20, USA).
For the measure of cell growth by optical density (OD)
at 620 nm, vegetative cells in liquid media were monitored
by reading turbidity at 620 nm (3) with a spectrophotometer
UV-VIS Metrolab 325 digital. One ml of the
standardized microbial suspension was added to different
kinds of media. Sterile media without microorganisms
served as control. The liquid media used was Mueller-
Hinton broth supplemented with different additives. Concentration
selection of all the components in each media
was made in relation to Mueller-Hinton labels, Dingman
and Stahly (4), and Gochnauer (8).
All the liquid media used and their respective composition
were identified by their initials 1) M: 0.2 % of Mueller-
Hinton broth dissolved in water; 2) M+T: 0.2% of Mueller-
Hinton broth supplemented with 0.1 mg/l of thiamine
(autoclaved separately); 3) M+Y: 0.2% of Mueller-Hinton
broth, 1.5% of yeast extract; 4) MYT: 0.2% of Mueller-
Hinton broth, 1.5% of yeast extract supplemented with
0.1 mg/l of thiamine; 5) MYPGP: 1% of Mueller-Hinton
broth, 1.5% of yeast extract, 0.2% of glucose, 0.3% of
K2HPO4 and 0.1% of sodium pyruvate; 6) MYPGP+T: 1%
of Mueller-Hinton broth, 1.5 % of yeast extract, 0.2% of
glucose, 0.3% of K2HPO4 and 0.1% of sodium pyruvate
supplemented with 0.1 mg/l of thiamine; 7) MYPGP without
pyruvate: 1% of Mueller-Hinton broth, 1.5% of yeast
extract, 0.2% of glucose and 0.3% of K2HPO4; 8) MYPGP
without glucose: 1% of Mueller-Hinton broth, 1.5% of yeast
extract, 0.3% of K2HPO4 and 0.1% of sodium pyruvate.
Triplicate analyses of each medium and strains were utilized
to determine the tests. The media were incubated
at 35±0.5 °C, for 24 hours.
Absorbance data from culture cells were statistically
analyzed as a randomized complete block design fitting
a model: media + strain and its interaction using SAS
PROC MIXED (SAS OnlineDoc (rtm). Version 8, Copyright
(c)2000, SAS Institute Inc). Least square means were
compared using the Tukey-Kramer test. The level of probability
used to assess statistical significance was a = 0.05.
The pH measurements of MYPGP liquid medium,
MYPGP liquid medium without sodium pyruvate and
MYPGP liquid medium without glucose were evaluated
in order to establish whether the absence of one of their
components influenced on pH and, hence, on cell
growth.
As shown in Table 1, acceptable growth was observed
on MYT broth, with major mean values of optical density,
compared to the other liquid medium, with the exception
of the Cobo strain. No significant differences were noticed
between MYT and the second medium with greater
absorbance values, except for the Cobo strain. For all
strains, poor growth in the MYPGP medium with thiamine
was observed in comparison with the MYPGP medium
alone; yet no significant differences (p < 0.05) between
these two media were noticed for all cases.
Table 1. Microbial growth expressed as optical density in different liquid media used
The MYPGP medium with no pyruvate yielded turbidimetry values of high absorbance when compared to those of the MYPGP liquid medium, even though these values showed no significant differences for all cases. The broth without glucose showed lower absorbance values (Table 2).
Table 2. Comparison between microbial growth in MYPGP liquid medium and same medium without glucose and pyruvate
against four P. larvae strains .
The pH values corresponding to the four strains studied
in the different media were measured, i.e MYPGP,
MYPGP without pyruvate and MYPGP without glucose.
Mean values were 6.11 for all the strains in the first broth,
6.14 for the second broth, and 6.422 in the MYPGP broth
for the MYPGP broth without glucose addition.
The indispensable components for P. larvae growth
are thiamine (B1 vitamin), yeast extract and several peptones.
Sporulation is promoted when glucose and sodium
pyruvate are added to the medium (4). Yeast extract
provides great variety of nitrogen organic constituents
capable of covering the general nitrogen needs
and growth factors usually required for microorganism
growth (14).
For all of the cases, poor growth was noticed in the
MYPGP liquid media with thiamine when compared to
MYPGP liquid media. Most likely, this is due to the addition
of this micronutrient concurrently with the nutrients of
the MYPGP liquid media. The additives can become either
growth inhibitors or toxic when their concentration is
high (14). Assays on the MYPGP liquid media without
glucose and without pyruvate in comparison with the
MYPGP liquid media were carried out afterwards (Table
2), so as to explain the lower absorbance detected in the
liquid MYPGP in comparison with MYT (Table 1). Findings
were consistent with the results published by
Dingman and Stahly (4), who stated that pyruvate would
inhibit growth and promote sporulation while glucose
would favour growth. The growth in these media was
evaluated separately, as it can be inhibited by organic
acids resulting from glucose fermentation. After these experiments,
we tested the selected MYT liquid medium in
assays with essential oils against strains of P. larvae. For
all the experiments, the MYT liquid medium yielded good
bacterial growth. Besides, the MIC values in studies of
antimicrobial activity could be determined by turbidimetry.
In view of the values of absorbance at 620 nm observed
in the MYT broth, which are above those of the
MYPGP liquid, it is concluded that the best media to evaluate
antimicrobial activity is this new enriched broth. Given
its nutritional quality, it favours good P. larvae growth;
and due to its translucent coloration, it enables bacterial
growth assessment by turbidity.
Acknowledgements: The authors would like to thank Dr. Pablo Zunino for critical review. Also Claudia Faverin and Adriana Cano for the assistance in providing the statistical analysis. This work was supported by UNMDP, ANPCyT and CONICET.
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Recibido: 23/04/07
Aceptado: 02/06/08