CHAPTER - II

7

REVIEW OF LITERATURE

The occurrence ot Shigellosis varied from

country to country and within a particular country over

a period of time (Christae, 1968). During 1969- 1 71,

Shigellosis appeared in the form of a pandemic in Central

America which affected six countries. In the early mid

1970's, Shiga bacillus was isolated from the epidemic in

Asia (Rahaman, 1984). In developing countries, children

between the ages ot one and four mostly suffered due to

Shigella infection (Boyd~ al., 1980). In developing

countries infants less than six months old and especially

neonates were infected with Shigella (WHO report, CDD/80:

1). In the last two decades there appeared reports of

dysentery outbreaks mainly caused by §· dysenteria~ I

throughout the world. Shigellosis was reported from the

countries in which socio-economic condition and sanita­

tion system were really very poor. It was reported from

Japan (1898, 1967), Central America (1968-'69), Mexico­

city (1972), Bangladesh (1972-'78), Stmartin Isldnd

(1973), Canada (1974), Costa Rica (1974, 1984-85),

Ethiopia (1974-'80), England and Wales (1974-'78),

Srilanka (1976-'82), Maldives (1982), Burma (1984-'85)

and in rn;ia from southern parts (1972-'78), Eastern

parts (1984-'85}, from Kashmir (1983) and Andaman &

Nicobar Islands (1986). Thus Shigellosis which showed

a global distribution (WHO report CDD : 80/1) was

mentioned in the Table ( 1 )

8

Bacillary dysentery due to §· flex~ri assumed

epidemic proportion in the USA in 1966 {Reller, 1970).

More than 16,000 cases were reported to the Centre for

disease control {Marr ~! al., 1980}. In the United

Kingdom, §. ~ei was the most dominant Shi~la species

in 1937 (Taylor, 1957). In an outbreak in Mexico-city in

1972, the strain carried the same plasmid cording CSSUT

{Chroramphenicol, Streptomycin, sulfonamide, Trimethoprim)

resistance and an another plasmid encoding for ampicillin

resistance character (Filroy et al., 1976). Donald~~~~.,

(1987) described the occurrence of the different subgroups

and serotypes of Sh~ella in the South-Western part of the

province of the Cape of Good Hope of the Republic of South

Africa during the period between 1?68 and 1985. In Johan­

nesburg (1951), 70% of Shigella isolates were Shigella

flexneri {Kahn ~! al., 1957) but in Durban, it was 74%

between 1960 and 1975 (Brachman et al., 1970; Rubidge

et ~., 1978). Shigellosis outbreak also occurred in

West Bengal, Tripura and Manipur af India in 1984 (Pal,

1984). Strains of Shigella resistant to various commonly

used antibiotics were earlier reported from various parts

of the world including India {Panda and Gupta, 1964;

Sharma~~ ~l·• ~967; Urban, 1972: Abraham et al., 1969;

Arya et al., 1977: Paniker et al., 1978;

9

Khan et al., 1979; Arora et al., 1981; Agarwal~ ~1_.,

1984) and in Bangladesh (Bennish et ~l·' 1985). Thus

the pandemic of bacillary dysentery which originated in

the Central American countries in 1969-'70 (Mata ~ al.,

1969; Gangarosa et al., 1970) due to appearence of highly

virulent strains of ~· dys~teriae type 1, spread to

widely different geographical areas of Asia and Africa.

Bangladesh (1972-'78) was the first country in south Asia

to be affected, followed by Southern India (1972-'73),

Srilanka (1976), Maldives (1982), Eastern India (1984),

Nepal (1984-'85), Bhutan (1984-'85), Burma (1984-'d5).

The pandemic was unprecedented fot its fast-spreading

capability and the severity of the disease characterised

by its high attack rate and fatality due to various

unusual complications.

The geographical regions affected by the

Shigellosis have been mentioned in this section for better

understanding of the global epidemiological trends. In

Central America, Shigellosis was first recognised in

Guatemala (Masley ~i al., 1966; Mata et al., 1969) which

affected hundreds of villages and many cities (Gangarosa

et al., 1970}. In 1967, ~· g~enteriae was isolated from

a patient who acquired it in Mexico and also from a

person who returned from Ethiopia (NCDC report 17, 1968;

Reller~~~~·' 1969). In respect of total isolation of

10

Shigellae in 1968, £. sonnei accounted for 54% and

s. flexneri for 44.3% as compared with 36.7% and 62% in

1964. Similar trend was witnessed in Great Britain,

Western Europe, Japan and Korea (Chun, 1964; Gillies,

1964; Aoki, 1968; Kostrzewski, 1968; Eichner et ~.,

1968) . (

The rapid spread throughout the region was due to

S. dysenterial I infection (Mata ~i al., 1969).

s. ~~enteriae was isolated from both non-hospitalised

villagers and hospitalized patients (Top~ al., 1968;

Scott ~~ ~·· 1968). Thus thi.s was the first documentary

country wide epidemic due to s. ~enteriae I in Western

hemisphe~e (Mata et al., 1969) though previously isolated

in Guatemala (Beck~~ al., 1957). Concentrations of

8 Shigellde reportedly did not exceed 10 bacilli/g (Dale

et al., 1968; Dupnot et ~.!.·• 1969; Caceres et al., 1970).

Epidemic §.higa bacillus showed that 52 out of

53, were resistant to Sulfathiazole, Chloramphenicol,

tetracycline. None was resistant to ampicillin, kanamycin,

colistin, gentamycin, cephalothin, Nalidixic acid, neomycin

or nitrofuratoxin. Most isolates were intermediate in

their susceptibility to penicillin and Erythromycin (Mata

et al., 1969). The mortality {8,300 deaths) was high ln

Guatemala (Mata et al., 1970; Gangarosa et al., 1971;

Morris~~~~·· 1971; Reller ~i ~!·• 1971).

11

Shigella isolated from Atlanta, Georgia, USA

(October 1967-'70) were resistant to streptomycin, tetra-

cycline, ampicillin and sulfonamides (Farrar ~tal., 1971;

Eidson et al., 1976). --were isolctted in New york city hospitals, USA. ~· ~£~i

was resistant. to ampicillin and tetracycline (Neu and

winter, 1973). s. ~~ei and§. flex~~ri 2a isolates

obtained from Ontario (Canada) were found to be resistant

to Ampicillin, tetracycline and trimethoprim. The emergence

of TMPlSMX-resistant strain was reported for the first

time in Ontario region in 1978 (Bannatyne et al., 1980).

In Brazil, §higell~ flexneri strains which were

isolctted in 1982, were found to be resistant to trimetho-

prim-sulfamethoxazole (TMP-SMX). In Southern Brazil,

Shigellae were isolated from 33 out of 34 patients

(Korzeniewski et al., 1984). In June 1982, an outbreak )

of gastro-intentinal complications caused by ~· !Onnei,

occurred in two places in Oklahoma. Swimming was consi-

dered as a potential source of infection there (Makintubee

et al., 1987).

An outbreak of dysentery due to §. dysentery

type 1 took place in a hospital-ward for children in Mexico

city in 1972. The strains were resistant to ampicillin,

Chloramphenicol, sulfonamides and susceptible to cephalo-

thin, colistin, Gentamycin, Kanamycin, trimethoprim and

12

Nalidixic acid (Olarte ~ 21:·, 1970, '71). On June 12,

1972, the epidemic occurred among the children of 5-months

to 4 years old and 5 out of 22 affected (Valera et al.,

1971; Galindo et al., 1973).

A strain of epidemic §· £~nteriae type 1

which was resistant to ampicillin, chloramphenicol and

tetracycline was also isolated from Costa Rica in January

1974 (Lizano et al., 1974). According to Crosal et al.,

(1985) ampicillin resistance might be considered as a

global problem of serious nature.

A wide spread outbreak of Shiga dysentery was

reported from Central Africa in 1980 (Rowe et 2l·' 1982;

Malengreau ~tal., 1983; Frost et al., 1985). Actually

it first started in North East Zaire and luter spread

through Rwanda (1982-'83) and Burundi (1981) (Frost et al.,

1985). Isolates were resistant to ampicillin, streptomy­

cin, Sulfonamides and tetracyclines. In 1981, nalidixic

acid w~s used for the treatment of Shigella and the out­

break was declined in Zaire (Malengreau ~~ ~~., 1983)

but one strain was still resistant to nalidixic acid

(R -- A c c S Su Sp T Tm Nx) and several to ·Kanamycin

in Rwanda. In all these epidemics 2· £~enteriae I strains,

the drug resistance was plasmid mediated except for

nalidixic acid (Frost et al., 1985).

13

During the epidemic of Shiga dysenteriae in

Zaire (1980-'83) there was noticed an important feature

i.e. the rapid adaptation of causative organism to changes

in antimicrobial therapy. The use ot co-trimoxazone resul-

ted in the spread of a strain carrying plasmid mediated

trimethoprim resistance. Trimethoprim resistant

~· 3ysenteriae type 1 was also reported from Bangladesh

(Zaman et ~~., 1983), the United Arab Emirates (Me Cormack,

1983), Kashmir (Panhotra and Desai, 1983) and west Bengal,

India (Pal, 1984).

Cahill~ al., (1966) reported an epidemic of

s. dysenteriae I in Johar of Somalia (Mero,E; 1976). About

240 ~· dysenteriae type 1 infections were isolated from

the patients suffering from dysentery during March 1973 -

October 1976 in Mogadishu and rural areas of somalia

(Mabadej, 1974). These epidemic strains were resistant

to ampicillin, chloramphenical, Kanamycin, Streptomycin,

tetracycline, trimethoprim, sulfonamide and Polymixin B

(Ribiczey and Beke, 1975; Tassew et al., 1982). During . 1968-'85, Shigellae were isoldted from 1562 patients

attending Tygerber hospital (Gadebon and Passew,1982;

Frost et al., 1985). According to Strule"Ms et al.,(1985)

Shigellaemia did occur rarely. In the opinion of Davis

et 2_1:_., (1976) 66% children died due to infection of

ampicillin sensitive straias.

14

In December, 1963, an outbreak of bacillary dysen-

tery caused by ~· gysenteriae 1 was reported from Itala and

·Giohar districts (Kevin et §l.·• 1966). The mortality rate

w a s 1 5% ( D u a 1 e , 1 9 61 ; Davey e t a l. , 1 9 61 ) . S hi. g e 11 o s is

thus appeared as a major health problem in Africa (Bardman,

et al., 1955; Philbrook et .21:.·• 1958; Bokkenheusor, v. 1959).

In 1984, 44% of Shigella spp were isolated from a childhood

dysentery epidemic in Thailand, from where s. flexneri and

s. ~nei were isolated (Taylor et ~l·• 1984).

The first nationwide epidemic of bacillary dysen-

tery in Tanzania, due to all Shiaella serogroups including ----a recently introduced multiple drug resistant Shiaella

£ysenteriae type 1 strain suddenly assumed alarming proper-

tion in November, 1981. It had been spread to oar es Salaam,

Ruvuma, Tanga and other parts in April, 1982. By the mid-

June, 1982, 12,423 cases with 125 deaths had. been reported

on the Tanzanian mainland (Mhalu et al., 1984).

In Ethiopia, multiple drug-resistance within

~higella genus (Gedebou S:.!. al., 1979) and within four

Shigella serogroups (Gebreyahannes et al., 1980) had been

reported. These were collected from January 1974 to Febru-

ary, 1980, {Gabr et al., 1984). Most common resistance

was TCA CbS Su (Frost et al., 1981). ---

15

273 Shigellae from Addis Ababa and 87 from its

rural surroundings were isolated and identified (Bauer et

al. , 198 3') • A multiple drug resistant .§.. flexner i 2a was

also isolated in Cape town (Gebre-Yohannes ~I ~l·' 1984).

~· dysenteriae I was not reported from Philippi-

nes but a few from Singapore, Thailand and Indonesia

(~mic health Statistics, 1986).

During 1972~'80, 3056 Shigella strains were

studied from fecal matter of patients in England and Wales.

Of these, 249 belonged to Shigella QY2~~ri~ I (Gross

e t ~!.., 19 79) • In Enaland, the incidence of s. sonnei ;J - ----

increased but that of .§.· flexneri decreased in 1977

(Morbidity & Mortality weekly report, HMSO, 1979). The

isolates were resistant to all common drugs including

Ampicillin (Grosset al., 1984; Row et al., 1984). Recently

.§ • .§.Onnei was isolated in London (Jarvis et al., 1984)

and the strains were sensitive to trimethoprim.

Jonsson et al., 1972 reported the occurrence of

Shigella infection to Roslagstull hospital,Stockholm,

Sweden. All the strains, except one were resistant to

Sulpha-isodimidine and streptomycin and other drugs

(Tunvall et al., 1984). s. sonnei and s. flexneri isolated

during 1970 in sweden were resistant to Sulfonamides,

tetracycline, chloramphenicol, ampicillin, neomycin and

16

Kanamycin (Urban, 1972).

During 1976-'78, 3552 strains of s. sonnei were

obtained fr~m czechoslovakia (Havlik, 1982; Hora et al.,

1982). 2· ~ei were resistant to azlocillin (Zdravo­

trieke listy, 1946; '77). An unknown serovar of ~hi~la

flexneri was isolated in Soviet Union also.

The first epidemic due to S. dysenteriae type 1

was reported in 1898 by Shiga in Japan as previously des-

cribed. According to Shiga, 89,400 cases with 22,300 dea-

ths were reported within one short period (Shjga, K 1898).

Multidrug resistant isoldtes were first noted in Japan in

the early 1950's (suzuki et ~l·, 1956). The majority of

the strains weie resistant to tetracycline, chloramphenicol,

streptomycin dnd sulfondmides (Mitsuhashi et ~~., 1969).

80% of Shigella strains were isolated in Japan in 1967

(Tanaka et ~!·, 1969).

In France, Rubinstein et al., (1971), reported

multiple drug resistance (TCA S Su) in s. fl~eri and I

s. sonnei strains.

During 1980-'81 there was a Shigellosis outbreak

in Malaysia (Jagathesan, 1984). The isolates mainly belong-

ed to ~· flexneri, ~· so~ei and rarely to ~· dysenteriae

serotype-3 (Bauer et al., 1966). Shigellae isolated in the

suburbs of Zhengshou city, China in 1981 1(Wei et al.,1984).

17

.§_. ~enteriae was the predominant serotype. All Shigellae

were sensitive to gentamycin, neomycin, aureomycin,

terramycin and Sulphadiazone.

In Polland, Shigella dysenteria~, ~· fl~ri

and other serotypes were isolated. They were multiresis­

tant (Noworyta et al., 1972).

Shigella outbreak occurred in Sudan in 1976,

caused by 2· £ysenteriae type 1 (Tassew ~ al., 1982).

The mortality rate was quite on the higher side (Hassan,

1985). The strains were resistant to ampicillin (Am),

tetracycline (Tc), Streptomycin (Sm), Penicillin,

sulphonamide (Su) but not to Chlor2mphenicol (Index

Medicus for WHO South-East Asia Regions; 1982-'83).

An extensive epidemic of Shigellosis due to

s. dysenteriae type 1 was reported from Banqladesh in

1972-'78. ~· dysenteriae was predominant type (Khan

et al., 1979; Khan and Shohidullah, 1980). Children

were ~ainly affected. The rate of mortality at Dhaka

hospital was 1~/o (Rahaman et ~l., 1983). s. dysenteria£

I and S. fle~~i accounted for 75-80% of the total

Shigella isolated from 1976-1981 (Rahaman ~~ al., 1983).

All ~- dysenteriae 1 strains became resistant to tetra­

cycline and Ampicillin (Mutanda et .§..1., 1981). The

epidemic strains of ~· g~nteri~ ~ype 1 were sensitive

to ampicillin and lacked 120 kb plasmicll (Ampicillin

18

resistant plasmid) (Pal Chaudhuri ~!. al., 1985).

s. £ysenteriae type 1 which was isolated from an epidemic

of Shigellosis in southern Bangladesh found to be resis­

tant to nalidixic acid, encoded by a conjugative 20 M

dal plasmid. This epidemic revealed an interesting pheno­

menon in as much as it showed resistance to nalidixic

acid - an antibiotic to which Shigellae were mostly

susceptible. Besides, the plasmids were previously

thought not to mediate resistance to nalidixic acid

(Munshi ~ al., 1987; Rogeric, 1986).

The epidemic broke out in St. Martin islands

during April-July, 1973. In St. Martin Island, Shigella

dysenteria~ I isolates were sensitive to ampicillin and

Kanamycin but resistant to tetracycline, chloramphenicol

and streptomycin (Khan~~~., 1975; Rahaman ~ ~~.,

1975; Levine et ~., 1973).

An epidemic of bacillary dysentery due to

s. £~nter i~ .1 had been reported for the first time in

Burma during Nov. 1984 - Feb. 1985 (Tin-Aye et ~~., 1984/

85). All these strains were sensitive to ampicillin,

cephalothin, gentamycin and Furazolidone but were resis­

tant to streptomycin, tetracycline, chloramphenicol and

sulfonamide (Tin Aye et ~~., 1985).

19

Epidemics of Shigellosis did also spread to

Nepal and Bhutan during 1984-'85. s. g~nteriae type 1

was isolated in Nepal but detailed report was ldcking.

§· g~enteri~ was also isolated from the shri-

gellae out break in Maldives islands s]nce April, 1982.

Strains were sensitive to nalidixic acid, gentamycin,

neomycin, kanamycin but resistant to commonly used anti-

biotics (Rezvi et al., 1982).

The pandemic of shigellosis due to ~· ~~enteria~

type 1 was also reported from Srilanka in April, 1976. It

first broke out in Jaffna and then spread to Colombo in

June, 1978. It was also reported from Kandy in May, 1972 \1

where it claimed 59 lives. Strains were resistant to

Ampicillin, tetracycline, Chloramphenicol, Furazolidone

and Co-trimoxazole but were only sensitive to nalidixic

acid and gentamycin. (G m). ( vel8uthapillai,1982).

An epidemic of bacillary dysentery due to

s. dysenteriae type 1 brofe out in Andaman Islands, India

during January-April, 1986. A total of 3057 cases with

28 deaths were reported in Port Blair (Sen~! al., 1986).

Shigella isolated in Delhi, India, between 1967 and 1971

were sensitive to neomycin and kanamycin (Kaliyu~rumal

et al., 1978). s. dysenteriae type 1 caused an out break

20

of the bacillary dysentery in a South Indian village in

1972 (Mathan ~! al., 1984). The strains were resistant

to ampicillin, chloramphenicol, streptomycin, tetracycline,

erythromycin but sensitive to neomycin, kanamycin etc.

Four localised outbreaks of similar nature were also repor­

ted from Tamil Nadu between 1973 and 1978 (Macaden et al.,

1980). Bhat et ~~., 1975 also reported a Shiga-dysentery

outbreak in Tamil Nadu in 1972. Isolation of a similar

multidrug-resistant 2· dysenteriae I strain from hospita­

lized patients was also reported from Karnataka during

1976-78 {Mathan et al., 1982). Bhat et ~., 1980 report­

ed a Shi~ ijacillary dysentery caused by§· £~nter~gi

Karnataka in 1980. Hundred strains of Shigella isolated

from Karnataka (Bangalore and Manipal) and Kerala (aalicut

and Trivandrum) during 1976-77 were studied. §.dysenteria~

I were resistant to ampicillin, chloramphenicol, sulfona­

mide, streptomycin and tetracycline (Paniker et al.,1978)

and resembled the ampicillin-resistant strain of Costa­

Rica (Virmala et ~~., 1972; Paniker et al., 1978; Khan

~~ ~., 1979; Lizano et al., 1979). An epidemic of 2hig~

dysentery was reported from Vellore which was caused by

s. dysenteriae I in -1983-'84 (Jadhav et al., 1985; Koshi

et al., 1985). They were resistant to all commonly used

drugs including ampicillin and nalidixic acid (6%).

21

It had already been mentioned earlier that Shiga

dysentery outbreak was reported from West Bengal (India)

from February, 1984 which finally spread to the south. A

team of scientists from NICED, calcutta, investigated this

Shig~ dysentery outbreak in Hooghly district (Pal, 1984).

The attack rate was 9.7% for all ages. Isolation rate

varied from 70-80% (Sen, 1983}. The drug-resistance

pattern of s. dysenteriae I was previously descrjbed. This

outbreak claimed 3.500 lives. Thus~· dysenteriae I·conti­

nued to be major aetiological agents of dysentery/gastro-

enteritis in India (Paniker et al., 1978; Agarwal ~ al.,

1981; Panhotra & Desai, 1983).

A global epidemiological trend of Shigellosis

was shown in the Figure ( p N E)(~ ) . The spread of

Shigellosis in India was pointed out in the Figure ( TW0)(2}

Ca~eful epidemiological observations on the

changing pattern of antibiotics resistant strains in Japan

disclosed several unusual clinical features during some

epidemics (Falkow, 1975). The individual patient contain-

ed both sensitive and multiple drug resistant Shiqella

strains of the same serological type. According to Akiba

et ~!_., (1960) it could be explained on the possibility

of multiple resistance being transferred from the drug

resistant E. coli to Shigella in the intestinal tracts

of patients. Ochia et al., (1959) also reported the

Fig. 1

Fig. 2

Map showing Shiga dysentery outbreaks

in different countries throughout the

world

Map showing the affected areas caused

by Shigella in Indian subcontinent.

_fft\_-T Ql.OBAL.

. ~~­.;...'t ~..,p -v:\'1 . ~

'1 -~~ -

_ ... .oo 2-

.-+ MAL DNES ( ·~g 2)· .!:l-"8'(7 'BAY OF ~'Et-t~Al. • U ~ L1C:.A NZ,A ( ''~~ ) • "R~ RWAN':DA t\'H1)

. • 33, -+ '8 R LIN"..l:>.J: ~ 9-n) .

• AA ~- A:DbiS_ A"B~~A ( I)H- t)!o) - ., '(fTHIOP1~).- ---• .l>L_. :r.~t.Hr. (e,p)

-~ -~-.;,.~---: ........ .,! .... - -. .... • .... -...--; ... ~

-- - ----··~··-- _..._ '"""~~- ~--- ~ .... n. ..... . .

E PIDEMIOL OGlCA L TRENDS OE SHia£ LLOSIS • + ..:BLACK ~QUARE£-+AFFECTEDAR.EAS

.... -·-. --.r~.>.- MfJN"'fJLIA• ,

:r-~ l:>AH"(') 8 '6) ..._,.._ I<OR~A.( ''&I)

~~,~~· ~,.~.llo,.,.., .... (u••·•>R~-Tm .. ,..,.. -~ -"lo . .. ... ) :J",.,o~~ ~ ·n,"'f~,_ . kE,.. :..,~l-4 ~ ... _ ,,_,~~ 1.-tf,.__ "'¥" ~.£-

·y-~-1 - ~ ~., ·~"" ( ~., . .,......,¥~ ... (,,.~) ... ,,~1 '~~ . ..,~~ ~31~1 ,. ¥'1 ........ '''"' ''~(' ...., V>~o., • ., """:~ ,. 'Ar.

~ 13

<!<' · '41o 1 ' ., · ~, 1-'J'.1t '/? ... ~.b -_, t, ~p 0~ ~~~ • •

....... ~,e "-~o<,,.~ !) ..

1--

~

Z· 0 ...... ~ z

Table 1 GLOBAL DISTRIBUTION OF SHIGELLOSIS

-----------------~----Country j_ Shigellae

---1. Central America S.sonnei (Predominant) 1968-''69 (Mata et al.1

2. Costa Rica

,, 3. Canada

4. Brazil (South America)

5. Mexico City

6. Ethiopia·

7. England & Wales

8. czechoslovakict

9. China

10. Malayasia

11. Japan

12. Sudan

13. Srilanka

14. Burma

S.flexneri 1969 -- --S~ ~ysenteriae 1 Gangarosal

197.0)

s. dysent~iae 1

s.flexneri Qal :s.sonr;er-S. flexneri

1974 & (Linzano 19::34-'85 et al. I 1974)

1974 (Piechaud et .£1·, 1974)

1982 (Tiemens et ~l· 11984

June, 1972 (Olarte

~.£ysenteriae 1 & other Shige 11 ae

e t ~!... 1 1 9 7 6)

1974-'80 (Gabrel Yohannes

et gl., 1983)

S.flexneri, S.sonnei 1974-'78 (Rowe et al., 1984)--& ~.dysenterTae ___ __

s.sonnei

~.£ysenteriae (Predominant) and other Shigell-ae

1976-'78 (Hora',K,V and Valvik1 J.l 1982)

1981 (Wei and Zhang et al., 1984)

S.flexneri, S.sonnei 1980-'81 and .§_.9_ysent~.E_iae--

(Jagathesan 1 M., 1984)

serotype 3

~.dysenteria~ 1 and other serotypes

§_.dysenteriae 1

1898 1950

1967

1976 & 1982

1976-'82

1984-'85

(K.Shiga,1898) (Suzuki et al.,

19 56) (Tanaka et al., 1969)

(Hassan, 1985)

(Val aut hap i 11 i 1

et §.l· I 1982)

(Tin Aye et al. 1

1984/85)-

Table ( ONE ) contd.

Country I Shigellae --~r __ Year~ __ R_e __ fe __ re __ n_ce ____ _

15. Bangladesh ~.dysenteriae I and 1972-'78 other shigellae

16~ St.Martin Isla~d ~.~enteriae 1 1973

17. Andaman and ~.dysenteriae 1 1986 Nicobar Islands·

18. Maldives Island ~.~enteriae 1 April, 1982

19. south India

a) Tamil Nadu s. dysenteri~ 1 1972-'78

b) Karnataka s. dysenteri~ 1 1976- 1 78

c) Kerala s. dysenteriae 1 1976-'77 (Calicut and Trivandrum)

(Rahaf)lan ~! al. 1

1983)

(Rahaman et al. 1

19 75}

(sen~D. etal.l 1986)

(Rezvi et al., 1982)

(Bha t et al. 1

19 75)

(Ma than et al. 1

1982)

(Paniker et al. 1

19 78)

d) Vel lore s. dysenteriae 1 July, 19831 (Koshi ~! al., J un e I 1 9 8 4 1 9 8 5 )

2 0. Eastern India

west Bengal s. dysenteriae 1 1984 (Pal,SC,1984} (In a vill.::3ge in Hoogh.ly district)

21. Other States

Kashmir A few Shigellae 1983 (Panhotra & isolated Desai, 1983)

~--------------

22

similar findings at a meeting of the Society of Chemothe-

rapy of Japan. Kagiwada et £l., (1960} observed that 60%

of the total hospitalized patients showed multiple resis-

tant Shigella in their stools. These results thus while

supporting in vitr£ experimental results of Akiba and

Ochiai {1959/60) revealed that the transfer of resistance

could occur between Shigella and ~· £Oli in the gut

(Falkow, 1975}. The modes of gene-transfer might be due

to either (a) transformation or (b) conjugation or (c)

transduction (Akiba and Ochia, 1960}.

The spread of transmissible drug resistant plas-

mids (R-plasmids) seemed to be responsible for Shiga

dysenteriae outbreak world-wide. In 1980, Shigella isola-

tes were reported to be resistant to combination of

trimethoprim and sulfamethoxazole (SXT) for the first time

(Bannatyne et ~l·• 1980; Taylor et al., 1990) and such a

resistance was according to Taylor et al., 1980; plasmid

mediated.

rl

When plasmids carry genes encoding antibiotic

resistance; they are called 'R-plasmids' or 'R-factors'

(Garrod, 197d). Mitsuhashi (1960) proposed the term

11 R-factor" for the transmissible resistance property

(Falkow, 1975). First 11 R-plasmid" was described in

Enterobacteriaceae by Watanabe {1963). The antibiotic

resistance of pvasmid encoded in the epidemic strains of

23

Shigella was first discovered in Japan in late 1950's

(Watanabe, 1963). In Japan, the first R-factor was isola-

te~ from Shigella sp in 1958, whi~h showed resistance to

tetracycline, streptomycin and chloramphenicol (Falkow,

1975). Olsen and Shipley (1973) observed the transmissi-

bility of R-plasmids to all genera of the Enterobacteri-

aceae. R-plasmids commonly showed resistance to Kanamycin,

Neomycin and paramycin (Benveniste and Davies, 1973).

Bacteria of medical importance acquired resistan-

ce to many clinically useful antibacterial drugs, within

a short time after new drugs were· introduced.

It was found that the R-factors conferrina resis-

tance on Sm, Cm, Tc and su (Sulfonomides) present in many

samples tested for epidemic strains of §. £ysenteriae I,

belonged to the incompatibility group 'O' plasmid (Datta

et~1:_., 1974).

In Shigell~ antibiotic resistance, though not a

part of virulence factor, it played a significant role in

the clinical management of disease (Olarte et al., 1981;

Timmis ~t ~., 1986). Antibiotic-resistance might be

chromosomal or plasmid mediated in 2· dysenteriae (Timmis

~ al., 1986). Like other bacterja in Shigella, the

chromosom71 and plasmid resistance bearing genes generally

24

expressed their effects in different ways : for chromo-

somal genes resistance was typically the result of an

alteration in ribosomal protein or RNA polymerase etc.

whereas the plasmid genes typically dictated the synthe-

sis of enzymes which in turn inactivated the antibiotics

pushed into the cell (Goodenough, 1978) or plasmid-

mediated resistance frequently involved enzymetic modi-

fication of the antibiotics as reported in Shi~lla

dysenteriae (Timmis et al., 1986.). some 'R-plasmids'

carry only one resistance gene while others carry two or

more •. Many plasmids carry genes that determine their

own infectivity or transmissibility between - bacteria

by conjugation {Hayes, 1969) as it was observed in

~higella and Salmonella (Falkow, 1975). At the end of

the second World war the sulfonamides or 'Su' were found

to be more effective against Sh.ig_~ causing outbreaks

in Japan. The introduction of Sm, Tc, Cm to Japan in

1950 was subsequently followed by their extensive use.

In 1956, Kitamato reported the isolation of §higell~

flex~eri strain resistant to Sm, Tc, em and Su.

A significant number of Shigella showing

multiple resistance bearing R-factors, were isolated .,

on a regular basis (Table 2· 3•4) I )

25

Table ( 2 ) Data showing the occurrence of

Year

antibiotic resistant Shigella strains in Japan

by Watanabe et al., 1960.

Numb Shio -----stra test

er of ella ins ed

sm Tc em Sm Sm, em & TC

____ ...... __ _ - ·----1953 4,900 5 2 0 0 0

1954 4,876 11 0 0 0 0

1955 5,327 4 0 0 0 0

1956 4,399 8 4 0 0 1

1957 4,873 13 46 0 2 37

19 58 6,563 18 20 0 7 193

1959 4,071 16 32 0 71 74

1960 3, 396 29 36 0 61 308

26

Table 3 ) Data showing resistant plasmids from multiply Resistant epidemic strains of Shigella £YE.enteriae type 1 (Modified version of Frost ~tal., 1981; The Lancet, Nov. 14, 1981).

Location Year(s) No. of Plasmid Resistance No. of strains compata transfer- st-rains examin- -bility red with ed group plasmid

Central America* 1969-72 7 B cs SuT 7

Bangladesh 1973 21 B CT ssu** 5 B T SSu** 4

II T 8

N T 4

India 1978 4 B CT ssu** 2 FI ACS SuT 2

Sri Lanka 1976 B CT SSu** 9

1978 9 B CT SSu** 11 12 FI ACS SuT 1

Somalia 1976 5 X ACT SSu** 3 X AT SSu** 2

FI me*** ACSSu'l' 1

Zaire 1981 6 X ACT 6

Mexico 1972 6 0 RCTSSu 7

Tanzania 1982 7 X RACSSuT Not men-tioned

Ethiopia 1980 6 X TCASSuCb -do-

* El Salvador, Guatamela and Mexico

** Independent ssu resistance determinant co-transferred with the group-B or Group X-plasmid

*** Present in the same strain as group X plasmid, AT S streptomyc~n/Sulphonamides resistance non-transferable A = Ampicillin, c = Chloramphenicol, s = Streptomycin, Su = Sulphonamid, T = Tetracyclin

Tab

le

( 4

; F

0 U

R)

Data

sh

ow

ing

an

tib

ioti

c resis

tan

t S

hig

ell

a

(19

85

) (afte

r

seam

ic H

ealt

h

Sta

tisti

cs,

19

86

)

Co

un

trie

s

Sero

var

No

. o

f N

o.

of

Nu

mb

er o

f str

ain

s

resis

tan

t to

M

ajo

r str

ain

s

res is-

I I

I j .

AB

FC Js

xT-r

esis

tan

ce

teste

d

tan

t C

p

TC

Sm

K

m

patt

ern

s

----

Ind

on

esia

s.

d

yste

riere

2

2 1

2 2

-2

-C

TS

A,

TS

A

s.

flex

neri

2

2 -

2 -

--

-T

Jap

an

s.

d

ysen

ter ia

e

2 2

2 2

1 -

1 -

CT

SA

, C

T

(To

ky

o)

s.

flex

neri

2

2

21

2

0

20

1

5

-1

6

11

C

TS

A,

CT

A

s.

bo

vd

ii

11

5

3 4

4 -

2 -

! TS

X,

TS

,

s.

so

nn

ei

69

4

3

12

3

6

42

1

9 1

5

I C

TS

A

---

Mala

ysi

a

s.

dy

sen

ter iae

2 2

1 1

1 -

2 -

A

• s.

flex

neri

9

0

65

5

4

57

5

8

2 5

5

11

C

TS

A

s.

bo

yd

ii

4 I

3 1

3 2

1 2

-C

TS

AX

Ab

bre

via

tio

ns

: C

p =

C

hlo

ram

ph

em

co

l,

Tc

= T

etr

acy

cli

ne,

Sm =

Str

ep

tom

ycin

, K

m =

Kan

am

ycin

AB

PC

=

Am

pic

illi

n,

SX

T

=

Tri

meth

op

rim

S

ulp

ham

eth

ax

afo

le.

An

tib

ioti

cs

sen

siv

ity

te

st

was

carrie

c b

y

ag

ar

dis

k d

iffu

sio

n

pro

ced

ure

. ~ ~

Transposons are descrete sequences of bacterial DNA

that can move from one replicon to another by a process

of recombination called Transposition or Translocation

(Froster, 1984). The transposable markers may be anti­

biotic resistance determinants~ toxins and other viru­

lence factors or metabolic properties (Cohen, 1976;

Klicknor, 1977; Nevers and Saedler, 1977; Kleckner,

1981; Calos and Miller, 1980; Starlinger, 1980).

Davidson et al., (1979) at the California Institute of

Technology found that the insertion elements play a

critical role in chromosomal gene transfer during bac­

terial mating. Molecular biologists are of the opini­

on that regions bound both insertion sequences and

repeated sequences of nucleotides at the end of anti­

biotic resistance elements can replicate independently

of other sequences. This leads to or spread of anti­

biotic resistance gene, enabling Shig~ll~ to become

resistant to antibiotics and thus enhancing their

pathogenecity (Heffron .~ al., 1975). This is how the

pathogenic resistance in Shigella gets transferred by

this way (Elwell et al., 1975; Phillips, 1976; Percival

et al., 1976).

Recent studies comprising the growth in

chemostat cultures of strains containing Tn 5 or Tn 10

with their nontransposon counter parts have suggested

29

that transposable elements may provide mutations which

provide a selective advantage to their host (Biel and

·Hart, 1933; Chao et al., 1983; Doolittle and Sapienza, --1980; Orgel and Rick, 1980). Tn 5 is an example of a

compound transposable element which contains an antibio-

tic resistance determinant to neomycin and Kanamycin (

(Berg et al., 1975; Jorgenson et ~l·• 1979; Rothstein

et ~l·' 1979; Black and Roth, 1980; Isberg et ~l·• 1982;

and Reznikoff, 1982). Transpos-:::>n de1~ivatives that

medidte operon fusions and inframe translation fusions

have become widely used tools for the study of regulated

genes in gram negative bacteria including Shigella

(Casadaban et al., 1977) Vanviet et ~., 1978; Bringer

et al., 1978; Harayama et al., 1980). The overall

frequency of transposition within a cell remained cons-

tant regardless of the number of copies of Tn 5 present

in that cell (Johnson et al., 19?4; Rothstein et al.,

1980; Lazaar and Syvanen, 1982). According to Grinter

(1983) there exists a broad host range cloning vector

based on Transposen Tn 7, which encodes TpR and SmR.

Transposen 917 was discovered by Tomich and Clewell

et al., (1980) as a plasmid encoded erythromycin resis­

tance (Emr) determinant (Miller et al., 1972; Susan

warster et ~l·' 1983; Krooset ~ ~l·' 1984; Way et al.,

1984).

30

The~e are four cldssic mechanisms of resis-

tance specified by plasmids in §hig~,

(i) Inactivation

,~ ( i i) Incompatibility

(iii) Bypass

(iv) Altered target site (Davies and Smith,1978)

"R-plasmids" may be transferred by conjugation

and this system of transfer is correlctted with the ferti­

lity inhibition and 'tra' operon in many gram negative

bacteria including Shigella. A conjugal 'R'-plasmid

usually contain a 'tra' operon. A major distinction

between the F and R-plasmids is that the 'tra' genes in

majority of the conjugal R-plasmids are highly repressed,

so that nctturally occurring R+ strains transfer their

plasmids only at a very low frequency. This repressed

phenotype known as fertility inhibition or fi+ may be

mutated to an fi "derepressed" state so that R-plasmid

is infectiously transmitted at high frequency. DNA-DNA

hybridization results indicate that all the F-like conju­

gative plasmids carry relctted fin 'O' sequences (Keatchye

cheah et al., 1987). It is also re1 JOrted that there

exists a physical relation of the fertility inhibition

gene, fin O, among the fin o+ and fin 0- (Mitra and

Palchaudhuri, 1984: Cheah and Skurry, 1986).

31

Conjugal R-plasmids in the fi+ and fi states

are clearly distinct from nonconjugative R-plasmids,

which are unable to transfer their DNA at all. A given

conjugal R-plasmid may be sepctrated into two smaller

plasmids, one containing the antibiotic resistance

genes (R-determinant component) and the other containing

th~ conjugal transfer genes (Resistance transfer or RTF)

(Hartley and Richmond, 1975; Broda, 1979).

In Shigella, R-factors have apparently evolved

by a classic recombination or, as in the case of ampici-

llin resistance, the insertion of transposons (Hedges

and Jacob, 1974; Heffron et al., 1977).

The molecular evidence suggests that plasmids

of incompatible functions have an overall relatedness

(Broda, 1979). In Shigella, like ~· coli, R-factor' s

chloramphenicol resistance is generally due to produc-

tion of chloramphenicol-acetylating enzymes, that

catalyses-acetylation of chloramphenicol in presence of

acetyl Co-A (Shaw et al., 1967; Suzuki et al., 1967;

Mise et al., 1968; Walker and Walker, 1970; Harwood

et al., 1971; .Benveniste and Davies, 1973; Dowding and

Davies, 1975; Gatfney ~~ ~l·• 1978).

32

R-factor mediated Penicillin~resistance.is

activated through amilase and penicillinase (B-lactamase)

in Shigella (Datta and Richmond, 1966: Egawa ~ al., 1967:

Jack~ al., 1970; Lindquist~~~., 1970; Kontomichalon,

1972; Dale et ~., 1974; Hedges~ ~l·' 1974).

11 About 85% of R-factors found among Shigella

isolates carried an 1 AP 1 gene (Yoma gashi et al., 1969;

swai ~~ al., 1968; '70; Lindstrom et ~l·' 1970). Strepto-

mycin phosphotransferase also inactivates streptomycin

(Yamada~~., 1968; Okamato and suzuki, 1970;).

Okamato and suzuki (1970) obtained an R-sm strain of ...

Shigella ~hich inactivates streptomycin in presence of

Mg+ions and ATF (Umezawa et al., 1967; Harwood, 1969;

Benveniste et al., 1970; Smith~~ al., 1970).

Kanamycin phosphotransferase enzyme of

Shigella phosphorylates Kanamycin, and neomycin {Okanishi

et al., 1967; Meda et al., 1968; Ozanne ~ al., 1969;

Benveniste et ~., 1971) Gentamycin adenyldte transferase

adenylates Gentamycin in Shigelle. (Martinet al.,1971;

Hedges ~i al., 1971; Benveniste et al., 1971; '73; sack,

1975; Nalin et al., 1975).

33

The mobility of the R-determinant components

and the ability of a given R-plasmid to 'pile on' large

numbers of resistance genes has major implications for

public health since R-plasmids can be transmitted not

only from cell to cell but also across species lines.

R-plasmids in Shigella vary in their stability (Dattat

1971).

Strike~~ al., (1987) have characterised the

drug-resistance plasmid NTP 16 (a multicopy nonconjuga­

tive 8.8 Kb Plasmid). Aoki et al., (1986) have analysed

two related R-plasmids.

1 some closely related plasmids are unable to

exist stably in the same strain, whereas others remain

stable. The former group are called incompatable pla.s­

mids and where several exhibit this property they are

assigned to the same incompatibility (Inc) group. The

latter group are called compatible plasmids (Broda, 1979).

Plasmid incompatibility is an automatic consequence of

the normal activities of certain plasmid maintenance and

replication functions rather than the province of any

specific 'Inc' gene (Novick, 1987). Incompatability

maybe 'symmetric' or 'Vectorial' (Richard~al., 1987)

Symmetric incompatibility is seen with coresident single

replicon that shares essential replication and maintenance

34

function and is due to inability to correct fluctuations

arising as a consequence of random selection of indivi-

dual copies for replication and partitioning events

within the plasmid pool (Ogura et al., 1983). vectorial ---incompatibility may result from interference with parti­.s

tioning (Nordstrom~t al., 1980) and has been observed

with cloned fragments of unknown function (Kumar et al.,

1985).

Incompatibility is an inherent characteristics

of all plasmids including Shigella and can be demonstra­

ted between homologons pldsmids (Projan ~ al., 1.985).

In Shigella, the incompatible plasmids usually share

more sequence homology than do the compatible plasmids

(Falkow, 1975; Garai et al., 1979; Shalita et al., 1980;

delacruz et al., 1980) and in the case of conjugative

plasmids 'sually specific similar transfer system

(Willetts and Skurry; Bradley, 1981; Datta, 1979).

There is a notable correlation between the incompatibi-

lity and conjugation group of pldsmids within a given

conjugation system usually belonging to a single incom-

patibility group (Willettes, 1984). Characterisation

of incompatibility is expressed by I replicon and . 2-replicon derivatives of the plasmids (Ryabchenko

et al., 1988).

35

The incidence of the R-factors in the total

Shigell~ populcttions varies throughout the world.

R-factors are the major source of drug-resistance-genes

in Shigell~ and Salmonella (Thorne et al., 1973). Types

of R-factors often ditfer from species to species as

well as from one geographical location to another

(Falkow, 1975).

Eight R-factors h~ve been described under four

compatibility groups distinct from F-like and !-like

groups. A strain of Shigella dysenteriae type 1 which

caused a wide spread outbreak of dysentery in Central

America carries a 'CSSuT' resistance factor (Mata et al.,

1970). The CSSUT F-factor designated as TP 125 is

incompatible with R-factors (Grindley et ~l·• 1972).

R-factors coding for inhibition of F-medidted fertility

are termed 'fi' (Watanabe et ~., 1964). some of the

non-I~li~ fi-plasmids hdve been included under compati­

bility group in shigella (Romero, 1970: Skurray et al.,

1987).

Gradual acquisition of drug resistance in

Shigella dysenteriae type 1 and other Shiaella spp is

an. important feature in the Shiga dysentery outbreaks.

Shiga bacillus which was initially drug sensitive in

Central America (El Salvador) and Mexico, acquired an

R-factor, R-Su-sm-Tc-Cm as the epidemic progressed.

36

Transfer of resistance from Shi~ell~ to E.££1!

which occurred in 107 of 112 strains demonstrated that

resistance was mediated by R-factors in Central America.

(Farrar et al., 1971). It has been reported that some

Central American epidemic strains of 2·dysenteriae type 1

contained R-factor which belonged to group 'O' (Thorne

et ~!.·, 1974).

Transfer of resistance from Shigell~ to ~.coli

K12 was greater in New yor1< city (Neu et ~!_., 1973). In

MeNican Shiga dysenteriae outbreak RCTSSU type of antibio­

tic pattern was reported (Olarte et al., 1971). In Mexico,

Galindo et al., (1973) transferred the drug resistance

character of 2· dysenteriae type 1 to drug sensitive

E.coli K12 strains and found two different plasmids in

~.coli, the first one responsible for resistance to chloram­

phenicol, tetracycline, streptomycin and sulfonamides

(Datta and Olarte, 1974). But ampicillin resistance­

character which was encoded by another one was reportedly

controlled by an unstable plasmid (Olarte et al., 1976;

Datta et al., 1973-'74).

4 Ampicillin resistance R-factor was transferred

from Mexican epidemic 2· dysenteriae I to ~.coli K12

strains and it was inferred that these strains were carry­

ing two different plasmids of which the 080 megadalton

plasmid that contained the ampicillin transpoon (Tn A)

sequence was nonconjugative (Crosa ~ ~., 1977).

37

Strains of Shiga bacillus with the same resistance pattern

coded by identical plasmids were also isolated in Costa

Rica and same strain with 5.5 megadalton plasmids were

found in Bangladesh (Olarte, 1981).

The emergence of TMP-SMX resistance in Shigella

sp was reported, for the first time, in Ontario of Canada

in 1978. (Bannatyne et al., 1980). Very close similari­

ties exist·in the patterns of drug resistance plasmids in

the epidemic strains of §. dysenteriae I obtained from

central Africa (Frost et al., 1982). The plasmid profiles

of strains isolated in Somalia have many similarities to

those of the strains of Zaire and the inc X plasmid

carried by the strains were indistinguishable. The SSU

plasmid in the Somalian strains was larger than that of

Zaire and incompatible with NTP 2 • Besides, it belongs

to the group ssu plasmids (Smith et al., 1974).

Like the inc X plasmids, the group II plasmids

coding for resistance to 'ACS Su Sp T Tm' were isolated in

Zaire and Ra~anda. The strains from srilanka, Somalia and

central Africa appeared to have 4-5 plasmids of smaller

size. Genetic studies showed that the Plasmids of srilankan

strains were same in size as that of central Africa· (Frost

et al., 1985). Both the central American and south Asian

strains carry plasmids belonging to incompatibility group

B, whereas those obtained from Central Africa carry group

38

'x' and IIplasmids (Frost~!~., 1982). All strains

of Central Africa were tested for resistance transferring

(Anderson and Threfall, 1974) .and transferred to E.coli

K12

(Jacob~ al., 1977). Non-auto-transferring SSU­

plasmids were tested for compatibility with SSU plasmids

which included NTP2

(Anderson et ~l·, 1968; Smith and

Anderson, 1974) and R 300 B (Barth and Grinter, 1974)

and RSF 1010 (Gridley and Anderson, 1972; Anderson and

Threlfall, 1974; Grinter and Barth, 1976). DNA was

prepared from transconjugants and transformants (Willshaw /

et al., 1979). Close similarities in the pattern of drug

resistance and cryptic plasmids in the epidemic strains

of Shigella dysente~ 1 in Central Africa (Vantreeck

et al., 1981) are noticeable.

In Zaire, some nalidixic acid resistance is

plasmid mediated (Garrod et al., 1981). The epidemic of

§higell~ in North East Zaire was caused by Shigella

dysenteriae type 1 which carried two factors for compati-

bility group 'x' (Rowe et al., 1979). The inc. 'x' plasmid

was same as in Somalia {Rowe et al., 1985). In Somalia, ---resistence to streptomycin and sulfonamides was not

transferred even by mobilization (Frost~ al., 1981).

In Tanzania,, during April, 1982, it was founp that the

group •x• ~asmid of Shigella dysenteriae type 1 encoded

resistance to Am, Cm, Sm, Su, Tc (R-type).

39

Nine drug resistance patterns were seen in

Ethiopia (Bauer et al., Gebre-Yohannes et al., 1983) in

§. dysenteriae 1. Here 'R-TCA Cb S Su' was most common

{Cb =carbenicillin). Frost et al., (1981) characterised

plasmid 'x' carrying ~· dysenteriae type 1, that encoded

resistance to TCA s su. In England, - resistance to ampi-

cillin was increased considerably in 1982. Trimethoprim

resistance was transferable and single plasmid mediated

(Rowe et al., 1984).

In sweden, R-factors detected were of common

origin, possibly from the R+ Shigella strains (Tunvall

et al., 1984). Resistance to streptomycin was not trans­

ferable by conjugation (Urban, 1972). The high frequency

of R-factors among the staff members suggested that

R-factors in bacteria were acquired by cross infection

(Johnsson et al., 1985).

In Bangladesh, Halder et ~~., (1984) reported

four common plasmids (on average approximating 140, 6, 4

and 1.94 M dal) from a large number of 2· ~senteri~ 1

strains isolated during April-October, 1983. An unusual

isolate of s. £~nteriae that contained three plasmids

having sizes of 200 kb, 3.7 kb, and 2T2 kb was an inter­

esting finding. The large 200 kb plasmid encoded for 4

HeLa cell invasion, 2.2 kb did not encode antibiotic

40

resistance, while 3.7 kb plasmid specified resistance to

Sm and Tm (Timmis et al., 1986). These strains showed

resistance to Sm, Tm, em and Tc. No plasmid location for

r r em and Tc was demonstrated (Higaki ~ al., unpublished

data). Cmr, Tcr determinants in these strains might have

r been introduced into their chromosome by fusion of a em '

Tcr - Inc •o• group plasmid (Timmis et al., 1986}. In

Bangladesh the strains of Shigella dysenteriae type 1

and Shigell~ flexneri were isolated from children suffer­

ing from Shigellosis (Haider et al., 1985). They found

multiple plasmid bands and in their opinion 38% of .the

strains transferred the drug resist~nce factor(s) to~

recipient ~· coli K12

• Where the plasmids in the mole­

cular weight ranged from 44-76 M dal were correlated with

drug resistance pattern. It was found that multiresistant

clinical isolates generally harboured a single large

transmissible plasmid. The strains isolated from asympto-

matic excreters demonstrated plasmid patterns different

from those isolated from children with Shigellosis although

the bands were relatively homogeneous wit~ each group

(Samadi et ~., 1985). Both the groups showed the presence

of a 140 M dal plasmid DNA conferring invasiveness and

such strains were positive to sereny test. This study

implied that shigella strains from asymptomatic excreters

also retained invasiveness. According to Jamieson et al.,

(1985) these strains contained 6.0 and 1.94 M dal plasmids A

41

of unknown function. In an epidemic of Shigellosis in

Southern Bangladesh, the causal organism, ~· dysenteriae

1 was resistant to nalidixic acid as previously mentioned.

The genes coding for resistance to nalidixic acid, but

not thoSe coding for resistance to ampicillin or co-trimo-

xazole, were located on a conjugative 20 megadalton plasmid

(Munshi and sac et al., 1987).

In New Delhi, (India) antimicrobial drug

resistance R-factors in Shigella were isolated during

1967 and 1971 which also showed their transferability

"" (Kaliyugaperumal et al., 1978). Recently, Am-resistant

S. dysenter-liae type 1 strains from a Shiga dysentery - '

outbreak in West Bengal, India in 1984-'85 revealed six

plasmids ranging from 120 kb to 2.5 kb (Palchaudhuri et al.,

1985). Positionally two large plasmids (120 kb and 70

kb) were above the chromosome, while the small plasmids

{10 kb, 6.5 kb, 4.5 kb and 2.5 kb) were below the

chromosome. Am-sensitive~· dysenteriae type 1 isolates

of Bangladesh lacked 120 kb plasmid; otherwise, they

showed similar plasmid patterns like west Bengal isolates.

Epidemic Shiga 1 strains of Assam and Tripura showed very

close resemblance with the plasmids of Bangladesh strains;

all these plasmids belonging to group Inc B.

42

~ •,

A comparative study of ECO RI fragments confirmed

the close similarities in the plasmid patterns in the above

mentioned strains and indicated a common origin. The

present author in collaboration with others of the Insti-

tute he belonged to characterised and compared the plasmid

patterns of §.. dysenteriae type 1, isolated from West

Bengal, India (1984); Burma (1985), Andaman and Nicobar

Islands (1986) and Costa Rica and Tanzania (1985-1987),

Bangladesh and many other countries. All these identifying

characters and patterns .of the plasmid profiles will be

described later on in the result section.

Though fresh isolates of Shiga dysenteriae

producing bacteria contain a large 120-140.M dal plasmid

but sometimes they are unstable and may be missing at

high frequencies. (Kopecko et al., 1980; Harris et al.,

1982; Sansonetti et al., 1982; Watanabe ~ al., 1985).

According to Maurelli et al., (1984) loss of the

large plasmid of §.. flexneri is accompanied by loss of

ability of bacterial colonies to absorb the dye congo-Red

Spontaneous deletion derivatives of the plasmid that do

not permit host bacteria to absorb Congo Red no longer

allow,them to invade HeLa cells (Sansonetti ~~ ~~., 1981;

'82). ~ild-type isolates of Shigella flexneri bind the

dye Congo Red, producing - red (Crb+) colonies but white

43

coloured mutants fail to bind the dye (Crb-) {Pyne et al.,

1987). It is reported that more than one genes were

needed for congored absorption, HeLa Cell invasion and

Sereny reaction {Maurelli ~ al., 1984).

Anthoney et al., {1985) isolated a series of

Tn 5 insertions in P W-R 100, the virulence plasmid of

Shigell~ flexneri serotype 5. These in~ertions demons-

trated that three separate ECOR 1 fragments of PW lOO

were req~ired for invasion of HeLa Cells {Formal~ al., ~

1985/86). The DNA-sequence of vir F, a locus associated

with virulence and the ability to bind Congo-red in

Shigella flexneri 2a that is located on a 140-megadalton

(230 kb) plasmid was determined and analysed (Saki et al.,

1986). Three proteins (30, 27 and 21 KD) were expressed

by 230 kb plasmid {Yoshikawa et al., 1986/87).

Large plasmid negative derivatives are

nonpathogenic in~· dysenteriae 1 (sereny, 1957).

Reportedly many heavy metals such as bismuth, lead,

boron, chromium etc. are plasmid - encoded (Summers

et ~~., 1978; 1984) in enteric bacteria (Albert, 1973;

Hedges et al., 1973; Smith ~ al., 1978; Willsky et al.,

1980; Tynecka et al., 1981). In Shigella, the metal

concentrations used was arsenate,- 400pg/ml and

44

sodium arsenite 1% (w/v) (Trevors et al., 1985). Much

earlier Hedges and Baumberg {1973) found that bacterial

resistance to arsenic compounds in Shigella was encoded

by a plasmid (R 773) transmissible arm:mg the strains

of E. coli. This plasmid conferred resistance to sodium

arsenate and arsenic trioxide {Smith et ~l·' 1978).

Such a Shigella strain was resistant to streptomycin and

tetracycline. Iron-metabolism in s. flexneri was

reported to be chromosomal mediated (Hale et al., 1984).

South and Ehrli.ch {1982) isolated a 3.2 kb plasmid

specifying resistance to Cadmium and inferred that this

plasmid arose from a large plasmid by independent

deletion events.

Another interesting observation by Tokuda et al., . --(1987) suggested that Na+ pump was encoded by a plasmid.

They studied the Napl-trans conjugants •.

Plasmid and chromosomal directed virulance

is multifactorial in s. dysenteriae 1 (Keusch et al.,

1973, '75; O'Brien stt..a.l,., 1977; Eiklid et al., 1983).

Both chromosomal and extrachromosomal segments that are I

essential for the expression of virulent phenotype have

been identified (Formal et al., 1983, '81). One aspect

of this phenotype, the invasion of colonic-epithelial

45

cells has its genetic components located on a large

120-140 megadalton plasmid found in all Shigella spp

(Sansonetti and Formal et al., 1982; Sansonetti and.

Oaks et al., 1985). Loss of the plasmid is accompanied

by the loss of invasive phenotype and inability to

produce Keratoconjunctivities in Guineapigs (Sereny

!j; al., 1955; Sansonetti et &·, 1982, '83, '85).

Reintroduction of the invasive plasmid into a plasmidfree·

avirulent Shigella strain restores the invasive property

{Sansonetti et al., 1985; Watanabe~~ al., 1986}.

In s. flexneri, atleast four virulence loci,

two of which are involved in •o• antigen biosynthesis

are borne on the chromosome. The capacity to invade and ~

multiply within ·lukaryotic cells is encoded by a large

plasmid but in s. sonnei •o• antigen biosynthesis is

specified by its large invasive plasmid (Hale ~~ al.,

1983; Watanabe, 1985). In~· fle~eri the picture is

not very clear but in §· dysenteriae, the interplay

of plasmid and chromosomal determinants of virulence is

seen in its most intricate form; when a single factor, .

the •o•-antigen polysaccharide of the LPs is determined

by the genes located on both the small plasmid and

chromosome (Cabello et al., 1979; Elwell~~~., 1980;

Timmis et al., 1984 and '86). Such a disposition of

virulence determinants on plasmids help these extrachro-

46

mosomal rings to spread through exchange among Shigellae

of the same habitats and finally promote the evolution

of pathogenecity with increasing virulence (Timmis ~ al.,

1986).

A small 6 M dal plasmid in §. dysenteriae I

strains (W 30864) encodes one or more functions for the

type specific antigen and bacterial virulence (Timmis

et al., 1983). It is also reported that a small viru­

lence plasmid (6 M dal} of ~· dysenteriae 1 strain enco-

des a 41,000 dalton protein involved in formation of

specific lipopolysaccharide side chaims of serotype 1

isolates (Timmis et al., 1984). S. sonnei strains which

lose the ability to penetrate HeLa cells are avirulent

in animal hosts (Formal et ~l·' 1982). Shigella LPS

may facilitate adhesion of bacteria to the colonic mucosa

(IZhar et al., 1982).

s. dysenteriae 1 strain (W 30864) from a case

of dysentery in Dhaka, Bangladesh showed that its 9kb

plasmid specifying for LPS biosynthesis (Timmis et ~l:_.,

1984/85; Watanabe et al., 1984). ~higella lacking this

plasmid failed to synthesize •o• antigen indicating thus

that 9kb plasmid of some $trains of s. dysenteriae I

specjfies '0' antigen biosynthesis and therefore plays

47

a key role in pathogenesis (Watanabe and Timmis et a1.,

1984). DNA isolated from a variety of Shigellae isolates

with a radioactive DNA-probe fragment carrying an inter­

nal region of the rfb gene, demonstrated the presence of

this gene in all §· ~senteri~ type 1 isolates examined,

in which, it was carried on 9 kb plasmid but not in any

other 2· dysenteriae serotype examined (Watanabe et al.,

1984). It may be inferred that in case of Shigella both

the plasmid rfb gene and the chromosomal rfb genes are

necessary for LPS biosynthesis (Timmis et al., 1986).

Similar findings have been reported by Hale ~:!:. al.,

1984; Ermako et al., 1988. The following table

(Table 5 ;FIVE ) shows the plasmid and chromosomal

loci syecifying virulence associated factors of Shigella

( T i mrn is e t ~ . , 1 9 8 6 ) .

Table (

Strains and plasmids/ Chromosome

5

48

Showing the plasmid and chromosomal loci specifying viruleneeassociated factors of Shi~lla.

Role in Patho­gPnesis

References

Ll Locus ,. Possible

or funct1on region

------------- ------- ----~

Plasmids

Large plasmid

Shigella + E. coli

140 M D Plasmid

s. flexneri 2a ------

140 M D Plasmid

s. flexneri 2a ------

140 M D Plasmid

s. flexneri 2a

:t,arge Plasmid

s. sonnei

( 120 M D) Large

Plasmid

s. sonnei

His

His;

Xyl-Rha;

Kcp

His

Xyl-Rha;

Kcp; LDC

Invasion of and mul tipl i­cation within epithelial cells

He La Not

Yes sansonetti et al., 1982; '81-.-

Hale & Formal invasion clearly 1986 Sereny (-) explained Ve

He La invasion sereny test {+) ve

He La invasion Sereny test (+) ve Congo red binding

0-antigen

biosynthesis

For expression of group D somatic antioen

- I

invade HeLa cell.

Yes Formal & Hale 1986

Yes Formal & Hale 1986

Yes Kopecko et al., T9so

Yes Sansonetti et al.,1981 TFrOrri -Mini Review, Formal et ~l·, 1986)

Table (

Pl~smids/ Chromosome

Locus or

region

) contd.

Possible function

49

Patho-genesis

-;trains an]

________ ,_. ________ _ Ro_l_e __ i_n-~Referenc._e_s __ _

140 M D PLASMID

S. flexneri

9 KB Plasmid

s. ~enteriae

6 M D Plasmid

~· dysenteriae

type 1

Chromosome

S. flexneri

Chromosome

S. flexner i

Chromosome

S. flexneri

his and Pro­regions

Pure E/ Kep

argmtl

endoces five outer membrane Poly peptide s-140K, 78K, 57K, 4 3 & 39 K

'O' antigen Yes biosynthesis (Playing a

key role)

encodes for type specific antigen and bacterial virulence

'O' antigen biosynthesis

Essential for sereny reactions

Essential for sereny reaction and other functions

Yes

Yes

yes

Yes

Watanabe e t al., T9s4-

Timmis . et al., 1983

Formal et al., 1970; 19661 Sansonetti et al.,1983

Formal et al., TI97f)

Formal e t a 1., 19 66 Hale et al., 1970

Thus, one of the consistent attrjbutes of virulent

strains of Shigella is the ability to synthesize a complete

lipopolysaccharide (LPS) somatic antigen and this LPS becomes

a crucial: vjrulence determinant in the digestive tract (IIale

and Formal, 1987).

50

Maurelli et al., (1984) found that expression

of virulence in Shigella spp is dependent on the tempe-

rature at which the bacteria are grown. When grown at I

37°c, strains of Shigella flexneri 2a, shi~!l:.~ sonnei,

and §higella dysenteriae type ~ were fully virulent and

invaded Henle cells. They also produced keratoconjunctivi­

tis in Guineapigs. When grown at 30°C, the bacteria

neither penetrated Henlecells nor produced conjunctivitis

in the ~ereny-test and were phenotypically avirulent.

Strains grown at 33°c were only partially invasive in

0 the Henle assay, whereas strains grown at 35 C were as

invasive as strains grown at 37°c. Using the Henle

cell assay, it was determined that the loss of ability

to penetrate epithelial cells was completely reversed

0 by shifting the growth temperature from 30 to 3 7 c.

Restoration of invasiveness after growth at 30°c required

protein synthesis (Pyne et al., 1977; Hale et al., 1981;

Nastichkin et al., 1988).

Morris et al., (1986) studied altogether 42-

Shigella and 29 ~-£Oli strains which were screened for

invasiveness in the 'sereny test' and for hybridization

with two recently described DNA probes for the plasmid

invasiveness. Both probes produced identical results.

All sereny positive strains were hybridjzed with both

DNA probes.

51

Several polypeptides encoded by the large

plasmid of s. fle~i are associated with the bacterial

outer membrane (Eiklid et al., 1983) which is consistent

with the nature of bacterial invasion of eukaryotic

cells i.e. its initial event constitutes a cell surface:

cell surface interaction. A DNA fragment containing

the invasive genes carried by the large plasmid of §£•

flexneri was recently closed (Maurelli ~ ~l·' 1984)

which must be considered sufficiently adv~nced to acce-

lerate identification of cell surface components invol-

ved in bacterial invasion. The Tn-5 transposon was

used to obtain defined insertion mutant derivatives of

Shigella strain that lacked the 9 kb plasmid. These 'O'

antigen negative mutants exhibited an ability to invade

HeLa cells but inability to provoke a pqsitive reaction

to sereny test. 'O' antigen of ~· 9ys~nteriae type 1

is an essential virulence factor (Watanabe ~i al., 1984;

Watanabe and Timmis, 1984). Timmis et al., 1986, proved

that one 8f the R-prime 'his' plasmids which contained

about 40 kb of chromosomal DNA, when introduced into

E. coli K12 caused the synthesis of LPS of ~- dysenteria~

type 1. The species s. flexneri comprise a serologi-

cally heterogeneous group of.dysentery bacilli whose

'O' antigens are polysaccharide -Lipid-A-polypeptide-· i -_

lipid-S-complexes strictly comparable in their gross

~tructural and biological properties to the analogous

antigens present in all the gram neqative bacilli of

52

the enterobacteriaceae (Simmon et al., 1971). The LPS

component of these antigens consists of two distinct

regions - common basal structure shared by all

§· flexne£! serotypes and 'O' specific side chains

that determine the serelogical specificity and cross

reactivity of the whole antigen (Simmon et al., 1987).

Using stanAard gene cloning procedures and ~· coli as

host, an ,pproximately 11 kb-DNA segment carrying the

~· dysenteriae type 1 rfb genes was cloned from R-prime

'his' plasmid into the PBR 322 vector. Altogether 5

mutants were obtained which produced normal s.dysenteriae

type 1 LPS in E. coli K 2

(Okazaki et al., 1962). The 1 --

first step in 'O' side chain formation was encoded by a

small 9 kb plasmid whereas all other steps were specified

by the chromosomal rfb determinants, as established

from transposon mutagenesis (Timrnis et al., 1986;

Hardy, 1987).

Gene-cloning studies of the cytotoxin for

characterisation of the Shiga toxin gene were also

carried out (Keusch et al., 1973; Federal register

part III and IV, 1983 and 1984; Timmis et al., 1985;

I 86) 0

53

Timmis et al., (1986) generated two series

of R-prime plasmids carrying either arg E or the asn-

chromosomal loci of ~· dysenteri~ 1. The gene for

Shiga toxin was thus closely linked to arg E on the

genome of s. dysenteriae type 1 strains {Timmis et al.,

1985).

A Tn 5 transposon mutagenesis studies on the

plasmids thus revealed that all insertions which inacti-

vated •o• antigen formation was clustered with a 500 bp

DNA segment and the rfb gene encoded a 41,000 dalton

protein (Puhler et ~!.·, 1984). Yoshikawa et al., (1986)

isolated many independent Tn 5 insertion mutants in 230

kb plasmid when they used Shigella flexneri 2a strains.

A few of these mutants were negative in expression of

four phenotypes conside~ed namely invasion into epithe-

lial cells, congored binding, Mouse Sereny test, and

inhibition of bacterial growth but majority were posi-

tive for all four phenotypes •

. Usina S. flexneri 2a (YsH 600) Sasakawa - - ------et al., (1986), isolated 304 independent Tn 5 insertion

mutants in the 230 - kilobase invasion plasmid. Among

the 304 insertions, 150 were negative in expression of

four phenotypes examined; - mouse sereny test, invasion

54

to epithelial cells, congored binding and inhibition of

bacterial growth. In this opinion, more than two gene

clusters existed within this region. Both were required

for expression of all four virulent phenotypes. Formal

et al., (1985) isolated the mutant strains which were

unable to enter epithelial cells. A hybrid strain was

developed by incorporating the xyl+ rha+ of E. coli K 12

into the Shigella genome which retained the ability to

invade epithelial cells (Formal et al., 1965).

It has already been described that the plasmid ~

may be lost spontaneously in many bacteria including

shigella. In those instances where instability or the

loss of the property is difficult to determine, the

bacteria can be treated with 'curinq agents'. Like

spontaneous instablity, the cured strains include changes

in colonial morphology, resistance to antimicrobial agents,

antibiotic production and various metabolic capabilities

(Costa et ~l·, 1980).

Genetically, curing of the bacterial plasmid

means the elimination of desirable plasmid(s) from the

strains by treating with different curing agents in order

to identify or characterise the specific plasmid(s).

Moreover, often it becomes useful to obtain a strain

free of plasmid DNA (Chandler et al., 1984). According

55

to Caro et al. {1982), in some organisms, the loss of a

plasmid may not provide sufficient evidence to conclude

that the trait is plasmid encoded, as because many plas-

mids can integrate into the bacterial chromosome

(Wickner et al., 1981).

Different curing agents which have been used

in Shigella and many other bacteria include intercala-

ting dyes such as Acridine organe (Hirota, 1960);

Ethydium-bromide (Bouanch3nd et al., 1969); Quinacrine

{Hahn and Ciak, 1971) and many others like - Acriflavin

(Mitsuhashi et al., 1961); Provalin (Meynell, ·1972);

Quin~crine (Hahn and Ciak, 1971); Chloroquine; Rifamycin

(Johnston and Richmond, 1970); Sarkomycin (Ikeda~.!;. al.,

1967); Conmermycin (Danislevskaya et al., 1980). Ethyl

methane sulphonate {Willette, 1967); Nitrosoguinidine

(Willetts, 1967); Mitomycin-C (Chakrabarty, 1972);

Thymidine starvation (Clowes et al., 1965); Atabrine

(sevage ana Yoshikawa, 1967); Sodium dodecyl sulfate

( 1 970) . 2+ 2+ ( . ) Tomoeda et a • , 1 , N1. and Co H1.rota, 1956 ;

NOvobiocin (Swartz & Me High, 1977); Penicillin (Lacey,

1375); Imipramine (fvlolnar et al., 1978) and Guanidine

hydrochloride {Costa et §1_., 1980)· Besides the above,

Heat (May~ al., 1964) and temperature (Carlton &

Brown, 1981; Palchaudhuri, 1985) have also been used.

According to Carlton and Brown (1981) the concentration

56

of a particular curing agent can vary considerably.

This depends qn the bacteria, efficiency and the mode

of action of curing agent. Acridine orange usually

causes the loss of entire plasmid(s). It is used for

plasmid curing in Shigella and ~al~ella.

Ethidium bromide has been extensively used

to cure plasmids in a wide variety of bacterial strains

including ~· dysenteriae type 1 and its other serotypes.

Bouanchaud et al., ( 1969) used the ethidi urn ---bromide to eliminate plasmids in antibiotic resistant

Enterobacteria. Curing with ethidium bromide ~as obser­

ved at a high frequency th.an with acridine dyes. The

mechanism of action of the acridine organe and ethidium

bromide seemed to be a preferential inhibition of plasmid

replication (Hahn and Korn, 1969). Many agents like

acridine orange, ethidium bromide, Mitomycin C had been

used in curing members of the enterobacteriaceae of this

plasmid F (Hirota 1960, Stouthamer et al., 1963).

Among antibiotics, Rifampicin (Hahn and Clark,

1976) has been used in curing plasmids from many gram

negative bacteria (Chandler, 1984; Bazzicaluppo and

Tocchini-Valentini, 1972). Recently, the coumarine-.

57

antibiotics (Me Hugh and swartz, 1977; Hooper ~~ al.,

1984) have been found to cure plasmid(s) from different

host bacteria (Gellert et al., 1976; sugino et al., 1977;

Weisser and Widemann, 1985).

Freezing thawing induced curing drug resistance

plasmids from bacteria (Calcott et al., 1983, '88) Mito-

mycin 'C' is known to cure·the plasmidi via a correspond-

ing hydroquinone p~thway (Rheinwald et al., 1973).

Plasmid - R (Resistance), F (sex factor) and F II incom-

patibility group which cannot be cuJ?...ed with intercala-

ting dyes, can be cured by SDS-treatment in E. coli K 12.

An attempt was also made to perform such an experiment

in Shigella (Tomoedo et al., 1968). Bleomycin was also

used as curing agent. some physical agents are also

able to cure the plasmids. Temperature curing has been

reported in Shigella and many other gram negative

bacteria. Elevated incubation temperature (5° to 7°C)

above the normal or optimal-growth-temperature- can be

employed as a curing method. According to Carlton arrl

0 Brown (1981) 43 C temperature can cure the plasmid in

which optimum growth temperature is 37°C. E. coli K 12

has also been cured of F-Plasmids at a temperature range

0 of 42 - 44 c.

58

Palchaudhuri et al., (1:985) cured the plasmid

(large plasmid) in ~· dysenteriae type 1 which became

0 unstable when grown above 3.7 c. Thus temperature curing

has been reported in Shigell~ and many other gram nega-

tive bacteria.

In 'Curing' experiments of the present study,

the plasmids have been cured of the epidemic strains of

§• dysenteriae type 1 by using different curing agents.

A 'plasmid free' strain was also developed by using

acridine organe.

Like other bacteria, Shigella also exchange

their genetic material by recombination which may be

transformafion o~ conjugation (cell to cell contact)

or transduction. Therefore experiments concerning

transfer of genetic material that are conducted by

simply mixing culture of the test bacterium with a

suitable recipient strain can produce progeny by any of

the above three processes (Brooks et al., 1978). Trans-

formation is generally employed when two nonccnjugative

plasmids are being tested or when a nonconjugative plasmid

is introduced into a recipient carrying a conjugative

plasmid (Hardy, 1987). Small (5-10 kb) plasmids are

known to be transformed very efficiently however larger

(30 kb) are transformed at . low frequencies (Lederberg

~ ~- 1 1974) o

59

In any case, however, it is clear now that

transformation is one of the best devices to transfer

especially the nonconjugative plasmids (Hardy, 1987).

But only a very few reports are available about the

transformation of Shigella, though reports on conjugation

ph~nomenon are plenty. A brief literature review can be

provided for understanding the transformation phenomenon

in Shigella and to correlate with experiments conducted

here.

In many bacteria, including Shigella transfor-

mation may be natural or artificial (Porter £i al., 1978),

chromosomal or plasmid mediated. Some of them are

naturally transformable and become competent at particular

stages (Lacks, 1977). In some cases, competence is depen-

dent on diffusible competence factors (Tomasz, 1969). In ~

others, competence depends on the presence of components

of the cell envelope such as membrane proteins {Zoon and

Scocca, 1975; Sox et ~1., 1979) or autolysin (Seto and

Tomanz, 1975). Ehrlich (1979) first reported that compe-

tent cultures of some bacteria could be transformed with

CCC plasmid DNA, though no such report was available in

Shigella; The control of expression of genetic transfor­

mation among naturally transformable bacteria is not

well understood (Morrison et al., 1987). Competence

60

is also associated with the introduction of (a) a small

set of new proteins (Morrison and Baker, 1979; Morrison,

1981), (b) temporary cessation of synthesis of most other

proteins and (c) appearence of a number of unusual cell

properties, including an effi'tient system for taking up

DNA and promoting genetic recomb~nation between that DNA

and the cell chr mosome of many gram-negative bacteria

including Shigella (Fox et al., 1964; Mccarty, 1980;

Ravin ~! al., 1980; Morrison et ~l·' 1982). In some

competence is controlled by a specific set of factors

(Guild, 1969; Porter, 1969; Hotchkiss et al., 1973) •.

The critical cell density of a growing culture is deter-

mined by a mechanism involving a secreted protein, compe-

tence factor (CF) acting as a Feed back signal sensitive

to the population level of the culture, when CF accumu-

lates to a certain level, it induces cells in the culture

to become competent (Tamasz, 1976). Competence termina-

tes after a short period and inhibitor of activator is

released (Morrison et al., 1983). In Shigella competence

represents a critical stage for transformation. pH is ~

also an important factor (Tirbay et al., 1973).

sometimes Shigella and many other bacteria

may not become naturally competent. These are then

treated with high concentrations of divalent cations to

transform artifically (Cohen et ~~., 1972; Cosloy and

Oishi, 1973). In Shigella ca 2+ -ion plays an important

role in the process of transformation.

61

Like other ~ram negative bacteria, in Shigella

transformation occurs through the following steps :-

(a) Binding of DNA (Plasmid) to the outside of the cell

(b) Transport of DNA across of cell envelope and (c)

establishment of the transforming DNA either as a repli-

con itself or by recombination with a resident replicon.

A number of gramnegativc bacteria including

§higella and Salmonella are naturally transformable by

plasmid DNA (Lovett et al., 1976; Le Blanc & Haswell, ·

1976; Ehrlich, 1977; Canosi et al., 1978; Gryezan et al.,

1978; sox et ~!·• 1979; Macrina et ~l·' 1978; saunders

& Guild, 1980; Barany & Tomasz, 1980; Good et al., 1981).

In most of the cases•, Shigella and other

bacteria take up heterospecific as well as homospecific

DNA (Tyssum ~!. al., 1971; Socca et ~·, 1974; Dougherty

et al., 1979). These bacteria contain the uptake site

for transformation, which is 5' - A A G T G C G T C A- 3'

(Danner e~ al., 1980). In some g~am negative bacteria ---including Shiegella certain conjugative plasmids may

prevent the bacteria to be transformed (Elwell et ~l·'

19 77: Smith et ~l_., 1979; Albritton

~i al., 1981). In Shigella and many other gram negative

bacteria, the frequencies of transformation by plasmid

DNA are lower than those with chromosomal DNA (Notani

et al., 1981/82; Groomkova, 1977).

62

Another interesting finding is that plasmid

but not chromosomal transformation can be further stimu-

lated 50 fold by the addition of 10 mM CaC1 2 (Gromkova

and Goodgal, 1977) in shigell~ and many other gram

negative bacteria. In Shigella, stimulation of plasmid

transformation by divalent cataions occurs depending on

whether the plasmid is circular or linear. 2+ ca trans-

formation occurs with homologous as well as heterologous

DNA '

Cohen et al., (1972) first used cac12

treatment

in E. coli transformation with plasmid DNA. Subsequently

it was applied to many of the gram-neg3tive bacteria

including ~higella, Salmonell~· ~· £Oli, Erwinia and

proteus (Cohen et ~l·• 1972; Lederberg ~~ ~l·' 1974:

Chakraborty et ~!., 1975; Kushner, 1978, Brown et al.,

'1979; Priets et al., 1979; Lacy and Sparks, 1979; Haque,

1979; Gantotti et ~!_., 1979; David et al., 1981.

Thus in Sh.!_gell~, like other gram negative

bacteria which have been transformed, - cac12

treatment

is a mandatory step. (Primrose et al., 1985). Though a

few gram nrgative bacteria are transformable without

cac1 2 shock, such incident is yet to be reported in

Shigella (Old et al., 1985).

63

Many factors includence the transformation

of gram negative bacteria including ~hi~la. It has been

found that E. coli cells and plasmid DNA interact produc-

tivity in an environment of calcium ions and low tempera­

a ture {0-5 C) and that a subse1:uent heat shock is important

{Primrose and Old, 1977, '85). The treatment with EDTA,

cacl 2 and different temperature shifts, affects signifi­

cantly less plasmid transformation of protoplasts than

plasmid and chromosomal transformation of competent

cells~~ , . '

The mechanism of DNA uptake by competent cells

and protoplasts has been reported in Shiaella and Salmo----~---- -----

~lla {Weston et al., 1981). Transformation of~· ££li,

Shiaella and Salmonella is dependent on the presence of

divalent cations, the efficiency of transformation varies

with the cation present ca 2 ~ Ba2+ > sr 2+ ~

(Weston et al., 1981). Increase in the concentration of

cac1 2 generally increase the transformation frequency

(Humphreys et al., 1979; weston ~~ al., 1981).

In the transformation experiment conducted

here a plasmid free strain of 'E. coli K 12 - C 600'

has been usrf=d as the recipient. The optimum concentra-

tion of divalent cations necessary to produce maximum

yields of transformante in ~· £Oli. C 600 vary (Brown,

64

• 1980). For this strain (E. £Oli C 600), the divalent

cation solutions (pH 6.0 - 7.5) improved transformation

frequencies. The more was the dilution of inoculum used,

the greater was the magnitude and duration of maximal

competence obtained (Brown et al., 1979).

Covalently closed circular (C C C) and open

circular (O C) plasmid DNA are taken up by ~· coli inclu­

ding C 600 (Cohen ~ ~l·, 19 72) and Shigella. In E. coli

K 12 C 600, transformation with linearized plasmid DNA

occurs with frequency which is about 100 fold lower than

with C C cor OC-DNA (Bergmans et a~., 1980; Cohen et al.,

198 2) •

According to Hoekstra et al., (1980) rec B+

cells can be transformed with linear DNA but the efficiency

is only 1~/o. Dose-response curve suggest that a single

plasmid DNA molecule is enough to produce one transfer-

mant (Mottes et ~.!_., 1979; Weston et al., 1979, '81),

though more plasmid DNA molecules bind tightly to the

outside of the competent cells (SabelniY.ov and Domaradsky,

1981; weston et al., 1981). u.v. irradiation of competent

cells of E. coli K 12 including C 600 brings about an

increase in the efficiency of transformation with plasmid

DNA. The phenomenon is called IPTE (increased in plasmid

transformation efficiency) and is dependent on the l

65

activated state of Rec A proteins (Vericat et al., 1988).

Further, it is also known that the cells which have the

transformability also depend on the inoculum size used

as starter. Modal cell volume and transformation frequen-

cy changed with similar periodicity throughout growth and

the highest transformability occurs when recipient cells

are in maximum volumes (Youngman ~~ al., 1983; Kyle~~~!·,

1984; saunder ~~ al., 1984; southschek Bauer, 1985; Conley

~ ~l·• 1986; Simon et al., 1986; Berth et ~l·• 1986;

Saunders e t a 1 • , 1 9 8 7 ) •

'There are very few inf:nrma\ .i.on 0bout the

transformation efficiency in .§.!2igella. The rate of trans­

formation efficienc:; depends on the cell density, heatshock,

plating media and DNA-concentration, etc. in many gram

negative bacteria (Saunder et al., 1987). Under optimum

conditions, only about 10- 3 - 10-4 of the plasmid UNA

molecules i~ a transformation mixture will give rise to

transformants (Weston et al., 1981; Hanhan ~~ al., 1983).

Greczan et ~l_., (1980) showed that j f the recipient

carries a homologous plasmid and if.the restriction cut

occurs within the homologous moiety than this same marker

transforms efficiently. An explanation for the poor

transformability of plasmid DNA mole~ule was provided by

Canosi et al., (1978). They concluded that specifjc

activity of plasmid DNA .in transformation was dependent

66

on the degree of oligomerization of the plasmid genome

(fvlotles et al., 1979; Canosi et al., 1981). The explana-

tion of the molecular events in transformation which

generates the requirement of oligomers has been presented

by devos ~l al., (1981) and Canosi ~ al., (1981).

Ter iele ~ al., ( 198 2) evolved a process in

which the entire DNA in the competent cells tried to find

homologous stretches in the recipient chromosome. In

Shi~lla such a phenomenon is yet to be reported.

Transformation of plasmid DNA from a nonconju-

gative plasmid into the appropriate strain carrying the

transporon was possible and the transformants were selected

on antibiotic plates (Hardy, KG. 1987).

'Plasmid-curing' does not appear to be a

reliable method for characterisation/identjfication of the

plasmid(s) in §· £ysente~ type 1, simply bec~use several

plasmids may be cured at a time. In the present investiga-• r•

tion attempts are made to transfer the smallest plasmid

(2.5 kb) of§. dysenteriae 1 to a plasmidless ~· coli

C 600 strain, after purification ~nd convetent cell

formation with an aim at determining the role of the

(2.5 kb) plasmid in the drug-resistance pattern and LPS

biosynthesis. The details about modalities will be given

later.

67

The conjugation is controlled by conjugative

plasmids which in size vary widely from each other. such

6 plasmids of only 17 x 10 daltons have been found in the

Enterobacteriaceae (Jacob et al., 1977). Conjugation

has been detected in many members of Enterobacteriaceae.

It is also reported in gram-positive bacteria (Meynell

et al., 1968; Clew~~ ~l·, 1981). The genetic control

of ~· coli K 12 conjugation mediated by F and F-like

plasmids is really complex (Achtman and Skurray, 1977:

Willetts and Skurray, 1980). The majority of the conju-

gative plasmids isolated from nature are repressed for

conjugal DNA transfer (Watanabe and Fukasavva, 1961;

Meyuel et al., 1968; Keat-chye-Cheah, 1987).

The study of conjugation is really very

important to understand the basis of plasmid transmissi-

bility because of the epidemiological importance especia-

lly in gram negative bacteria (Pal Broda, 1979).

Conjugative plasmids of gram-negative bacteria

synthesize an extracellular organelle called a pilus which

is essenti~l for recognition of recipient cells and forma­fl

tion of mating pairs with them. In addition, the Pili

provide the site for adsorption of certain bacteriophages

which may be plasmid or pilus specific (Willetts, 1984).

There are five lines of evidence for sex pili having a

role in conjugation (Ou and Anderson, 1970). Actually

their precise role is not understood (Broda, 1979).

One suggestion is that pili serve as conduction tubes

(Brinton, 1971). Another is that they bring matinq

cells toaether (Curtiss, 1969; Hohn, et al., 1969). ~ -- --

Retraction does occur after treatment with heat or

cyanide (Novotny and Fives-Taylor, 1974, '78). Like

other bacteria in Shioella ~lso it is found that at ------higher densities, mating complexes composed of more than

two cells (Collins and Broda, 1975; Actman ~~ al., 1978).

The aggregates that are observed microscopically in

mating mixtures, atleast involving Hfr donors, may be

largely the result of growth and division, followed by

failure to dissociate (Broda and collins, 1978). When

an F-like and !-like plasmids are present in the same

cell, each species its own type of pilus (Lawn ~! al.,

1971).

There is no transfer inhibition between plas-

mids (Lawn ~1 ~l·• 1967; Romero et al., 1971) and comple-

mentation does not occur between transfer defective

mutants (Cooke et al., 1970; Willettes, 1970; Brodt

et al., 1974).

69

Contact formation presumably generates a

signal for the synthesis or activation of enzymes invol­

ved in the transfer of DNA; perhaps including the nicking

of F to provide a linear structure since HFr strains

always transfer from F, with a definite polarity. such

a site (Ori 'T' : Willetts et ~l·• 1975) has been mapped

genetically (Willetts 1972; Clarke et al., 1977, '79).

A number of plasmid belonging to several incompatibility

groups have related transfer systems to that of F

(Froster and iHllets, 1977; Achtman et al., 1978).

Hfr transfer is regarded as F-self transfer,

in which the bacterial chromosome is inserted to F

(Bachman et al., 1976). The conjugative plasmids

control the formation of sex pilus for pair formation,

transfer of plasmid; and they have a mechanism of

'surface exclusion' that reduces the conjugal efficiency

(Thomas, 1981). Surface exclusion seldom completely

prevents the isolation of atleast some transconjugants

in the test cross (Hedges, 1982; Hardy, 1987).

Genetic-analysis suggest that there are

different groups controlling the conjugation system

such as Inc F (Welletts and Skurray, 1980), Inc P

(Barth et ~l·• 1978; stokes et al., 1981) and Inc P-10

(Carrigan and Fillai, 1979; Moore and Pillai, 1982).

Though many naturally occurring plasmids are nonconjuga-

70

tive, still an ori 'T' site on the nonconjuqative 4

plasmids allows their transfer when they are present

in the same cell. The most common nonconjugative plas-

mids are relatively small (2-10 kb) but many of them are

efficiently mobilized by some coexisting conjugative

pla.smids. All these plasmids have mobilization genes

that probably initiate.transfer from their ori 'T' sites

and substitute for similar functions encoded by the

conjugative plasmid (Willetts ~~ ~l·, 1980).

Only some conjugative plasmids mobilize a

specific nonconjugative plasmid. It is suggested that

these conjugative pldsmids (derepressed) may be inc P,

Inc I and Inc F-type (Kleckner et al., 1977; Me !nitre,

1978).

Many other conjugative pldsmids mobilize

the chromosome by carrying a transposable DNA sequence

(Falen and Toussaint, 1976; Denarie et al., 1977; Haas --and Holloway,1978; Toussaint~~ ~l·, 1981; Van Gijsegem

and Toussaint, 1982). Many conjugative plasmids have a

brodd host range and mobilize the chromosomes of many

gram negative bacteria (Holloway, 1979) including

Shigella. Many other plasmids such as FP plasmids can

mobilize the chromosome (Franke et al., 1978).

71

1 With respect to DNA transfer, conjugative

plctsmid is passed from Donor (F+) to recipient (F-)

during conjugation (Gross and Caro, 1966; Cohen et al.,

1968; Flanders et al., 1968; Ohki and Tomizania, 1968; --Rupp et al., 1968; Vielmetter, et ~l·' 1968; Vapneck

and Rupp, 1970-'71; Kingsman and Willets, 1978; Bernard

et al., 1983).

In Sta£bylococci and many other gram negative

bacteria, a plasmid of approximately· 34.5- 35 kb was

conjugative and it mobilized nonconjugative plasmids

(Me Donnell, 1983; Townsend et ~l·• 1986; Udo and

Townsend et ~~·· 1987). According to Lederberg (1978)

conjugative gene transfer was unidirectional. For

constructing enterobacterial F-derived Hfr strains it

was necessary to transpose Tn 2301, a transposon cons-

truct in which entire F-transfer region hctd been cloned

into an ampicillin resistance transposon, from a plasmid

to the bacterial chromosome (Johnson and Willetts, 1980).

The origin of 'trans' (Ori T) is the sequence

within which conjugal transfer of plasmid DNA is initia-

ted and is absolutely in 'CIS' for plasmid mobilization.

All conjugative and mobilize plasmids so far studied

encode transacting proteins and contain a specific cis -

acting site, the origin of transfer (ori T) at which

72

transfer is initiated (Clarke and Warren, 1979: Willetts

' and Wilkins, 1984; Everett and Willets, 1982). In the

F - plasmid, ori 'T' is the site, at which the plasmid

DNA is ni!f:ked prior to unidirectonal transfer of a

single DNA - strand from Donor to recipient cell

(Willetts and Wilkins, 1984; Willettes and Manle, 1986).

It has been previously· mentioned that plasmid borne

fertility inhibition (fin) genes control the expression

of many plasmid-conjugntion system. Inc F plasmids have

two genes, fin '0' and fin 'P', the products of which

prevent transcription of tra J gene (Finnegan and

Willetts, 1973: Willetts, 1977).

'F' is an - atypical plasmid as it carries

no fin 'O' gene and for this reason it expresses its

conjugation system constitutively. Fin 'O' product can

be provided in 'trans' by other inc F plasmids, reducing

F-transfer by 100-1000 fold and ·making the cells resistant

to F-specific phages. such plasmids are called Fi+ (F)

which indicate that they are the~selves inc F plasmids

compatible with F. Fi- (F) include the remaining all

other group Cheah et ~., 1987. Among the F-like plas­

mids, RI and RlOO are repressed due to their production

of active fin op systems (Hirota et al., 1962; Meywell

and Datta, 1967). Other plasmids such F and Col v2 -

K94

are naturally derepressed for transfer (Timmis et al.,

73

1978}; Dempsey and Me intire, 1933; Cheah & skurry, 1986).

Altogether 13 transfer (tra) genes hctve been identified.

They are subsequently ordered by deletion mapping A

(Ohtsubo, 1970; ) . More recently

some other genes have been described (tra M : Achtman

et al., 1979, traN, tra U, tra V and tra W : Miki et ~.,

1978;· traY: Willetts and Me Intire, 1978). Sharp

et al., (1972) by means of heteroduplex method has been

able to correlate the genetical and physical maps. All

the 'tra' genes are contained within 62 - 93.2 kilobase

region of 'F' (Ohtsubo, 1970; Willetts, 1971; Davidson.

et al., 1975; Skurry et ~., 1976b; Kennedy~~ al., 1977;

Achtman et al., 1978; Willetts and Me Intire, 1978)

using deletion mapping, Chim~eras and -transducing

phages carrying parts of tra regions. While working on

the incompatibility grouping of plasmids, Datta (1977)

tested unknown R-plasmid against standard plctsmids of

known incompatibjlity group. It has already been des-

cribed about the drug resistance character and their

transferability· in the epidemic ~trains of Shiga bacillus.

It is reported that in multi resistant Shiaella

dysenteriae I strains of Costa Rica, plasmids are indep-

endent of others and c~n he transferred separately from

the multiple drug resistance factor (MDR+). In conjuga-

tion system, these strains are resistant to ampicillin,

chloramphenicol, tetracycline, sulfonamides, kanamycin

and carbenicelin when they are used as donors and

74

~· £Oli K12 as recipients. In conjugation, Ampicillin

resistant plasmid was transferred with MDR+ plasmid in

_§hi~la transconjugants carrying this ampicillin resis-

tant plasmid showed the frequency of about 1/1000 similar

to that of the MDR+ plasmid (Penarand~ et ~., 1976).

The plasmids of different Shioella strains were characte-_.....___ ris~d foll~wing the process of transconjugation. s.sonnei

had two plasmids in common : one was a self-transmissible

Fi+ plasmid of 46M dal encoding tetracycline resistance,

other was a 5.5 M dal Nonconjugative plasmid encoding

resistance to streptomycin and sulfa-furazole. Many

cryptic plasmids of 1.0 to 24.5 M dal were also present

in the different strains of Shiaella. Mobilization of

the 5.5 M dal streptomycin and sulfafurazole plasmid

and a 1.0 M dal Cryptic plctsmid was reported with all

six~- sonnei isolates during conjugation. According to

Jamieson et al., 1979, such a mobilization was mediated

by the 46 M dal self transmissible Fi+ R-plasmid and a

24.5 M dal Fi plasmid.

Kaliyugaperumal et ~l·• (1978) showed that

many strains of Shiaella dys~teria~ type 1 isolated in

Delhi, India in 1967, • 71, transferred sulpha triad,

streptomycin, chloramphenicol and ~mpicillin resistance

to sensitive recipient ~· coli K12

strain in vitro, which

indicated that the level of resistance conferred by an

R-factor depends on both the R-factor and host cell.

75

In the opinion of Lerman ~i al., (1973) many

multidrug resistant s. flexneri 2a strains were able to ----transfer atleast part of their resistancP to sensitive

E. coli K12

F, which was thus made resistant to 120 pg/ml

of sodium azide in mixed culture. Nalidixic acid resis-

tance plasmid of ~· fl~eri 2a strains was nontransferable

to E. £Oli K12 during conjugation. Presence of atleast

three different transmissible resistance factors was

reported in Shigella. The resistance plasmid of epi¢emic

shiga bacillus strains isolated in Dar Es salaam, Tanzania

showed that they were conjugally transferable to E. coli

K12 either partially or wholly (Mhalu ~! ~l_., 1984).

The plasmids of Mexican strains of Shigella dysenteriae

type 1 were transferred to ~· £2li K12 (w-1485) strains.

It concluded that the recipient received two different

pldsmids : one encoding chloramphenicol, tetracycline,

streptomycin and sulfonamide resistance but other encoding

only ampicillin character. R-factor encoding chloramphe-

nicol, streptomycin, tetracycline and sulfonamides was

of compatibility group 'o' in s. £ysenteria~ type 1

{Olarte et al., 19 76) •

. Salzman~! al., (1967) observed a case of

S. sonne~ wh±ch was resistant to sulphonamides, strepto-

mycin, tetracycline and chloramphenicol. Resistance to

these antibiotics was transferable by an R-factor. A

strain of .§. • .§Q!l!lei infected with an R-factor causing

76

resistance to Kanamycin manifested a high degree of

in vitro resistance to the drug when studied by conjuga-

tion process. According to Thorne et £!·' (1973) R

factors (R-C SSUT) of the epidemic Shiga bacillus strain

were introduced into an ~· coli recipient and remarked

that the levels of resistance to antibiotics and heavy

metals were identical except that the §_hi~la R-factors

provided slightly greater resistance to Chloramphenicol.

f· coli harbouring 'Shigella R factors' were found to

have- approximately 1,000 fold higher frequencies of

transfer into drug-sensitive ~· coli and s. !Y£hi.

Transfer of TMP-SMX (Trimethoprim-sulfametho-

xazole-resistance) resistance from §· flexneri to two

nalidixic acid resistant f· coli K12 , 185 NXr and 711

r (Lac-28, his 51, tryp-30, Pro - c 23, Phe Nx ) was

done by conjugation on nitrocellulose filter papers.

§higella strains transferred TMP-SMX resistance to

-8 -9 E. coli 185 NX at a frequency of 10 and 10 • One

transferred resistance to E. coli 711 at a frequency ot

10-10 • The transferable TMP-SMX resistance of the two

s. flexneri was associdted with three pldsmids of

different sizes, which sugc;ested that TMP-SMX resistance

did not originate from a single plasmid (Tiemens et al., ·

1984).

77

Tanaka et al., ( 1983) studied the distribution

of R-plasmids and the conjugal transfer ability of drug

resistance in Shigella strains in Japan by various mixed

culture methods. E. coli CML 4902 (r-, m-, Nar) was

used as the recipient of R-plasmids. To confirm the

resistance patterns of R-plasmids and their transmissi­

bility, E. coli CML 403 (r-, m-, rifr - resistance to

rifamycin) was used as the second recipient. E. coli

K12 MLI 410 (F-tet+ C tet, tetracycline) and T-Kam+

·1 strains were used for the mobilisation of nontransmissi-

ble-resistance in Shigella. The strains were resistant

to tetracycline, chloramphenicol, streptomycin and

sulfonamide. The conjugal transferability of these

resistant strains were tested. 97.5% stra~ns transferred

their resistance by membrane filter-method. The drug

resistance of many strains was mobilized by the presence

of F-tet or T-kan plasmid.

The extensive genetic homology of Shigell~

and E. coli facilitate the conjugal transfer qnd integra-

tion of chromosomal materictl from E. coli Hrf strains

to Shigella flexneri (Brenner et al., 1973; Formal~ al.,

1982). 140 M dal plasmid (Virulence factor) of Shigella

flexneri was transferred to ~- coli K12 first and seg­

ments of s. flexneri. Chromosomal material were then

transferred to the same strain. The virulence of these

transconjugant hybrids was assessed in the HeLa cell

78

model. Derivatives of an ~· coli K12

strains was

constructed by the stepwise-conjugal transfer of the

above mentioned plasmid to contain the virulence d~ter-

minants for full pathogenecity in vitro (Formal et ~.,

1982). Chinault, et al., (1986) reported that three

plasmids encodina resistance to antibiotics, including

the combination of trimethoprim and sulfarnethoxazole,

were isolated from E. coli following conjugative transfer

from.§· ..flexneri 2a strains. One of the plasmids

(PC N.I) was shown by subcloning and DNA sequencing

to carry a gene encoding d trimethoprim-insensitive

dihydrofq~late-reductase identical to that of §· £~)i

transposon 7 (Hollingshead and Vapneck, 1985). A second

plasmid (PCN 2 ) inactivate the streptomycin by a phospho-

transferase mechanism and also to encode sulfonamides.

There was a third plasmid playing a significant role

in plasmid mobilization.

Formal et al., (1986) used a mobilizing

plasmid from s. fle~ri serotype 5 into E. coli:_ K 12

by conjugation process. The hybrid - transconjugant

could invade mammalian cell in vitro (Sansonetti et al., ---1983). This invasive hybrid was then considered as a

recipient for chromosomal genes conjugally transferred

from s. flexneri 2a. Transfer of these 'his' and 'pro'-

chromosomal region from s. flexneri 2a in K 12 hybrids,

79

caused the expression of 2a somatic antigen by· the

hybrids. The hybrids could invade the mucosa of rabbit

ideal loop but failed to induce the fluid accumulation

(Formal et al., 1974). 140M d. plasmid DNA of

s. flexneri 5 was also cloned into the expression

vector gt 11 and showed that this pldsmid encoded

atleast five outer membrane polypeptides - 140 K, 78K,

57 K, 43 K and 39 K (Formal et al., 1971); Buysse~~~~.,

(1982) and developed a strain by intergeneric conjuga-

tion and transduction. Conjugation experiment was

performed between various E. coli K 12 Hfr strains and

~hiyella flexneri 2a virulent recipients and latter

reciprocJl transduction analysis with phage PI vir~ was done in order to establish a locus on the genome

of s. flexneri 2a, which seemed to be necessary for

its penetration of epithelial cells.

The plasmid responsible for form I antigen-

synthesis in s. sonnei could be conjugally transferred

to gal ~· salmonella ~~i strain. serological studies

indicated that the derivative strain produced the form

I antigen in addition to the normal ~· ~phi somatic

antigen (Formal et ~l·• 1981). GriffJths et al., (1985)

transferred one of the chromosomal segments (associated

with the virulence of ~· flexneri) to E. ~oli K12 by

80

conjugation and inferred that this segment coded for

the production of aeroba.ctin and for the synthesis of

an iron-regulated 76,000 daltton (76K) outer membrane

protein. Analysis of vari:::ms E. coli K12

- .§· flexneri

showed that the genes involved with th~ synthesis of

aerobactin and with the pr0duction of 76 K protein

\vere linked to the mtl region of s. flexneri chromosome.

{Carrano, 1979/84 WHO Scientific working group, 1980).

The present author has conjugally transferred

r a 120 kb (Large Am ) plasmid of the epidemic strain

Shiaella £ysenteriae type 1 to a nonpathogenic plasmid­

less E. £2!! K 12 KL 318 strain. Altogether sjx charac­

ters were identlfied in the 120 kb plasmid of Shioella

£~enter iae type 1. These Am-Nal transconj ugant showed

their resistance to em, Tc, Am and HeLa cell invasion.

Kerato obnjunctivities (positive sereny test) in guinea-

pigs. . r

It had already been described that em transcon~

j ugant carrying a 120 kb plasmid of _§. Qy_senter iae 1

s . became em after 7-8 subculture on Tryptic soy agar

plcttes (NICE D; Ann. Report, 1987).