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Effect of amino acids as growth stimulant for recovery of Shigella spp.

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Abstract

Key words: Response; Amino acid; Shigella spp.; Enrichment DOI: http://dx.doi.org/10.3329/dujbs.v20i2.8981 DUJBS 2011; 20(2): 201-204
Dhaka Univ. J. Biol. Sci. 20(2):201-204,2011 (Juty) - Short communication
EFFECT OF AMINO ACIDS AS GROWTH STIMULANT
rOR RECOVERY OF SHIGELLA SPP.
AsHragul Amur, LurrE Ana, ANowARA BEGUM* AND sine.Iul Isleira Kueru
Depnrtmerit of Microbiology, Llniaersity of Dhaka, Dhaka-1000, Bangladesh
Key words: Response, Amino acid, Shigeila spp., Enrichment
Shigellosis is the most virulent and serious, due to its invasive character, system;c
manifestations, se\rere nutritionai impact and tendency to become recurrent over
prolonged period.rtt In Bangladesh, among the routine hospitalized diarrhoeal patients,
the isolation rate of shigellae are approxim ately 72o/o.Qt S. flexneri ancl S. dysenteriae type 1
are the two predominant serotypes isolated in Bangladesh as also in other developing
countries. S. flenreri alone constituted about 6A% of the shigellosis (ICDDR,B 19E0), the
number described to 42'k in 1984.6) The presenc e of Shigelln is almost never been sought
in routine water testing.(4) J'his might be due to several factors including stress or injury
of cells, viable but non-culturable state, or constrains of routine biological media for the
isolation of Shigella.rs) In the aquatic environment, adverse environmental condition e.g.,
starvation or exPosure to sublethal temperature, salinity or toxic chemicals may stress
bacteria, which are capable of growth in warm blooded animal. Because of such sublethal
iniury or stress, bacteria may enter a viable but non-cuiturable conclition, which has been
defined by Nilsonto) as an inabiiity of cells to produce colonies on solid media even
following prolonged incubation. Shigella spp. have also been found to enter sucir viable
but non-cuiturable state in laboratory condition. I{ecently Colwell et al.(n has shown that
viable but non-culturabie cells of S. dysenteriae type 1 retained several virulence factors
and remained potentially virulent, possing a public health problem. Thus, the potential
public health hazard initiated by Shigella species existing in non-culturable state may be
significant. Conventional bacteriological culture media/methods are inaclequate to detect
sucir cells and special resuscitation methods are requiredo. The cells might re-grow when
the environmentai conditions become favourable for growth.rs) This study envisages to
assess the influence of certain essential amino acids as growth stimulants leading to the
recovery of stressed Shigella spp. in conventional culture media following newiy
designed pre-enrichment and enrichment media.
The foilowing amino acids were examined as growth stimulants e.g., Dl,*alanine, L-
alanine, l.-arginine, L-aspartic acid, l,-cystein, L-glutamic acid, glycine, L-histidine, L-
lysine, L-tryptophane aud L-tyrosine. l'he chemically clefined basal medium (CDM) rvas
designed to culture and recovery of Shigella spp. in this study. The compositions of CDM
*Corresponding author. <anowaraTl@yahoo.com>.
202 .{1.-\M ef ,tl.
were (g/l): mannitol,5.0; glucosg 2.0; NHaCl,0.2; ICHPOa,2.0; KHzPO+,1.0; MgSO+.7HzO,
0.2; CaClz.2HzO, 0.1 and MnSOr.HzO,10.0 mg; Ferric citrate, 5.0 mg; ZnSOs.7tbO,2.0 mg;
CuSOe, 1.0 mg; Na-molybdate, 0.5 m& agar 20.0 g and water 1.0 lit. After sterilization of
the above mentioned ingredients for CDM, the filter sterilized amino acid (0.001 fiml)
was added. In this study, S. dysenteriae type 1 (strain nos.279L4 and 9860) and S. flexneri
(strain nos.27450 and 27682) were used. The survival pattem of Shigella spp. was carried
out in 99 ml sterile phosphate buffer inoculated with 1. ml of bacterial suspension, which
resulted in the final concentration of approximately 2.76 " 10s cells/ml. The flasks were
then incubated at room temperature (25"C). All counts were done on nutrient agar, XLD,
MacConkey's agar as standard and the CDM. The number of Shigella cells were
decreased from day 1" to day 5 gradually and recovery rate on nutrient agar, XLD,
MacConkey and CDM were almost same because here we used the known inoculum of
Shigella cells for survival study. After 5 days, the cells of Shigella spp. decreased
drastically and became non-culturable at 9th day and did not grow on NA, XLD
MacConkey agar media except CDM containing different amino acids. So, we concluded
that amino acids had some influence for resuscitation of viable but non-culturable cells of
Shigella spp.
Culturable cells were,counted at0, 1,,2,4,8 and24 hrs and then at consecutive days
until the bacteria were no longer culturable. Two days after the apparent "die-off",5.0 ml
of sample was removed from each of the microcosm and transferred to 45 ml of having
decreasing strength e.g., U2, 1.15, U10, L/50 and U1.00 of the pre-enrichment media. The
composition of pre-enrichment media was (gfl): glucose 5.0; peptone 5.0; yeast extract 5.0;
IOHPOa 2,0; KHzPO+ 1.0; NaCl L.0; Na-pyruvate 0.50; Na-glutamate 0.50; arginine 0.25;
trytophane 0.50 and distilled water 1.0 L Plate counting was performed at every 6 hrs
interval according to drop plate method using nutrient agar, XLD, MacConkey and
CDM.
The newly formulated chemically defined medium supported excellent growth of S.
dysenteriae and S, flexneri and was to response to various amino acids. Both the strain of S,
dysenteriae and S. flexneri were found to preferentially utilize L-glutamic acid. L-arginine
was significantly used by S. dysenteriae but S. flexneri showed good growth while in case
of L-aspartic acid, they showed opposite response. The growth of S. flexneri was good
with L-alanine and L-cysteine but response was poor with S. dysenteriae. No significant
growth response was observed with Dl-alanine, glycine, L-histidine, L-lysine, L-tyrosine,
only S. flexneri 27450 showed good growth in presence of lysine. Both the strain of
Shigella spp. responded well with tryptophane. Response of Shigella spp. to different
amino acids has been described in Table 1.
Viable cell number of Shigella decreased sharply in phosphate buffered saline
indicating a stressed condition due to lack of nutrients. The initial count of 5.54 x lQs/ml
decreased after nine days there appeared no culturable cells in the microcosm (data not
EII'I]CI.OT AMINO ACII)S r\S CRo\4,,].ri S'IIIV{[]I,ANT IroR IIICOVEI{Y 203
shown)' Nutrit-'nt stressed cells oI Shigettn spp. were successfully recoverecl by thc newly
designed enrichment rredia frorrr its nor-r-cr.rlturable stage indir:ating apparent
Iesr-rscitaijon' Within 'lA - 72l-rrs viable count of ShigelLn reachecl r-rp to a nurnber of about
7.95 x 707 cfulml.
'I'able 1' Response of shigella spp. to certain essential amino acids in the chemically definecl medium
(CDM).
Amino acids
Shigellt spp. 11
10
S. tlr1senterine typei (9860)
S" dusenterine typel (27974)
5..flen;eri (27682)
S. ]icxreri (27450)
I = l)[.-alanine, 2 " L-alaninc, 3.= [.-arginir,,e, 4 .. l.-aspartic acic], 5 - i.-cysteine, 6.. [.-g11tar..ic t.rcicl,
7-. qit,cine. S = L-h:stidine, 9: l.-lysint, 10. l.-trvptophane, 1l .= L,, tyrosir.re.
- " nvo grrlivth, 1'r; = Iair gr-or'vth, I " Ileasonabl),'goocl growth. i-.r-: Coocl growih, ri-i = Excellcnt growti-1.
With thr: airn to lormulate the pre-enrichment ancl cnrichment culture mcciia that ca.
rccovcr SliiTiello cells froln ettvironmental samplt-.s, the response oi 11 amilo acicls was
exallineci irr the newli' designed chemicaily defined medillm. Complex media e.g.
nr-ttrienl agar or plate count agar (I'CA) \4'ere not Llsecl becallse these coniain various
orgallic t-tt-ttrietrts, gro',vth factors, vitamins and amino acids etc,, those are alreacly
preselrf in the meclia rn4'rich may nrask or prociuce artifact in the response study. S.
tirlsetttcrinc r'r'as lourrd prcierortiaily to utilize L-glutamic acicl arrcl L-arginine follo,,r,ed by
L'-aspartic acici aird L-alanir-re. 1'he response of S. JTerne ri to the aclciition oI L-glutamic
acicl and l--aspartic acid r'r'as significant. 'l'l-re rclatively less growth response was notecl
n,ith i--.rrElininr. ancl L,-alanine. It is eyiclent lrom ihis study that the.se amino acids are
esserriial for the g,row'th of S. drysanreriue ard. S. J\L:xrtcri but probably the,v lhemsi:lves
catlnot synlhesize them So, if lhese ;inrino acids are sr-rpplementeci in the growth
nrediut-n, r'r'hich might bc re'paired the stressed Shigeltn and enables their recovery on
isolat.ion mt:clia ioJlolving t:nrichrnc.nt. 'i'he addition of amino acids that caused p.,ririrr"
response rnigl-rt be r-rsefr-rl in tr-re Iormrlati^g nerv media trrat nlalz e.rich or isorate
strcssed or injurcci cells'5' 'i'he amino acicls lvhich shorved rnirrimal response may n.i be
essc.tial to incorporate into the mcdia to reco\rer the i^jured cells.
In this stuciv, atter11pls lt'eri-'ntucle to stimuiate il-re nutrient stressecl conditions.
Viable c--oiitlt of S. flcrttt:ri arrct S. dilsurterinc typel cells irr the phosphate buflered saii^e
(PI3S) irl micrcri'-osnls decreased sharplv lnciicatirrg a stresseci i:r:r-rclition d,e t' lack of
grolr.,th promoting nlrtrients,
(+) , { i.+ +
(, ) (+) (r) r,-f (+)
1. -r .r. +.r- (n)
1" t. 1..f t.i- _l-
(+) + +++ ++ (+)
(+) + J-++ ++ ti.)
+ ++ ++ t-++ ++
+ ++ ++ +++ ++
+++
+++
+++
+++
78
204 r\LAM s/ 41.
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ResusL:itati,rn au.1 r'it-'.rlcr,',ce tort'ar,1s t-nice ol viabic br-rt nonculturablt: Vibrio oulnifictLs
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Ilet:over"y of culturability of ar-r l-lOCl--str:esscci popr.rlation of [,schcricfuin coli after inr:ubation in phosphate btrffer: t'csr-tscitatiot'l oi regrol^rlh? r\pp1
  • S Dukan
Dukan S, Y l-evi ancl l) 'J'ouati 1997. Ilet:over"y of culturability of ar-r l-lOCl--str:esscci popr.rlation of [,schcricfuin coli after inr:ubation in phosphate btrffer: t'csr-tscitatiot'l oi regrol^rlh? r\pp1.