"~ Journal of ETHNO

PHARMACOLOGY

E L S E V I E R Journal of Ethnopharmacology 45 (1995) 141-147

Prophylactic therapy of Salmonella typhi septicemia in mice with a traditionally prescribed crude drug formulation

Y o u v r a j R. S o h n i * , P a d m a j a K a i m a l , R a n j a n M. B h a t t l

Microbiology Department, CHM College (University of Bombay), District Thane 421003, India

Received 14 November 1994; accepted 15 November 1994

Abstract

A crude drug formulation comprised of eight medicinal herb extracts was studied for in vitro and in vivo effect against Salmonella typhi. The formulation displayed inhibitory action against the test organism in vitro. Subsequently mice were challenged with a virulent strain of Salmonella typhi (Ty2) and the protective effect of the formulation was evaluated with post-infective, pre-infective, single and multiple dose schedules, administered either orally or sub- cutaneously. A schedule that included multiple divided doses prophylactically administered had a significant therapeu- tic effect.

Keywords: Salmonella typhi; Crude drug; Septicemia; Medicinal herbs

1. Introduction

Typhoid fever remains an important cause of ill- ness with the annual number of cases in excess of 12.5 million (Edelman and Levine, 1986). A major impediment to the effective chemotherapy of typhoid is the ever-increasing numbers of resistant strains of Salmonella typhi (Anderson and Smith, 1972; Goldstein et al., 1986; Eykyn and Williams, 1987). Novel antimicrobial agents are being sought

* Corresponding author, Department of Biology, Alabama A and M University, Normal, AL 35762, USA. Tel.: (205) 851 5329; Fax: (205) 851 5905; e-mail: aamyou01 @asnaam.asn.net.

~ Current address: Biochemistry Department, Institute of Science, Madam Cama Road, Bombay 400020, India.

to combat this problem and also for use in cases of relapses and in prevention of chronic intestinal carriage of the organism (DuPont, 1993). Medici- nal herbs represent a rich source from which novel antibacterial chemotherapeutic agents could be obtained. A large segment of the world population, especially in developing countries, depends on the traditional systems of medicine for therapeutic agents for a variety of diseases. In the current paper, a formulation comprising eight medicinal herbal ex- tracts has been evaluated for its action on Salmo- nella typhi both in vitro as well as in mice. In the traditional system of medicine in India, Ayurveda, the formulation is prescribed for a variety of af- flictions including intestinal disorders (Parashar, 1961). The formulation is composed of the fol-

0378-8741/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0378-8741(94)01207-G

142 Y.R. Sohni et al. / Journal of Ethnopharmacology 45 (1995) 141-147

lowing medicinal herbs: Boerhavia diffusa L. (Nyctagineceae), Tinopora cordifolia Miers (Men- ispermaceae), Berberis aristata DC (Berberidace- ae), Terminalia chebula Retz. (Combretaceae), Zingiber officinale Roscoe (Zingiberaceae), Azadirachta indica Juss. (Meliaceae), Trichosan- thes dioica Roxb. (Cucurbitaceae) and Picrorrhiza kurrooa Benth. (Scrophulariaceae).

2. Materials and methods

The dried and powdered parts of the constituent plants were bought from an authentic dealer in ayurvedic herbs (Ganesh Aushadhi Bhandar, Kalbadevi, Bombay, India). The following plant parts were used to make the formulation: Boer- havia diffusa (roots), Tinopora cordifolia (vine), Berberis aristata (roots), Terminalia chebula (fruits), Zingiber officinale (rhizome), Azadirachta indica (bark), Trichosanthes dioica (vine) and Picrorrhiza kurrooa (roots).

2. I. Extraction All the constituent plant powders in the for-

mulation were individually weighed and mixed in equal proportions. Extraction was carried out in 80% aqueous ethanol at room temperature (10 g/100 ml). A batch of 10 g for extraction contained 1.25 g of the constituent powders. The extract was completely dried in a lyophilizer. The dried extract was weighed and dissolved in ethanol (1 g/100 ml, w/v). This 1% stock solution was used to prepare dilutions for the in vitro susceptibility tests. In ad- dition to the extraction of the whole formulation, each of the constituent plant powders was separately extracted in order to conduct the in vitro antibacterial tests with the individual plant extracts.

2.2. In vitro susceptibility tests 2.2.1. Susceptibility. Susceptibility of the test or-

ganism to the individual extracts and the whole formulation was determined by employing the standard disc diffusion technique (Barry and Thornsberry, 1991). The zones of inhibition, if any, induced by the extracts on the growth of Sal- monella typhi were calculated.

2.2.2. Determination of the minimum inhibitory concentration (MIC). The extracts demonstrating

a zone of inhibition on the test organism were taken up for determining the MIC by the standard agar dilution method (Sahm and Washington, 1991). The stock solution was used to obtain the required range of concentrations. Since the con- centration of the stock solution was 1 g/100 ml (1%), a 1:10 dilution of the stock solution resulted in a 1 mg/ml (0.1%) concentration. Thus a series of drug concentrations ranging from 1-9 mg/ml (0.1%-0.9% of the stock solution) were prepared by adding different volumes of concentrated stock solution to nutrient medium. The media were then seeded with freshly grown culture of the organism (1 x 106 CFU/ml).

2.3. In vivo tests Swiss albino mice weighing between 18-22 g

maintained on standard pelleted diet and water were used in all in vivo tests. Mice were employed in groups of 20 animals for each experiment.

2.3.1. LDso. Prior to the onset of the protection tests in mice, the LDs0 of the test organism was determined according to the method of Reed and Muench (1938). A virulent culture of Salmonella typhi strain Ty2 (obtained from Haffkine Research Institute, Bombay, India) with phosphate buffered saline (PBS) as the vehicle, was injected in- traperitoneally (i.p.) into sets of mice in increasing concentrations. The LDs0 was calculated as 1 × 104 CFU in PBS (detail not shown).

2.4. Challenge dose For the protection tests, mice were challenged

with a 100 × LDs0 dose of 1 x 106 CFU of Sal- monella typhi in PBS, injected i.p. in an inoculum of 0.25 ml. The survival of mice was examined until 2 weeks after bacterial inoculation.

2.5. Test drug administration The drug formulation was administered as a

whole preparation to the mice. The animals were administered the drug in two modes. One set of the animals was given the drug orally (p.o.) while the second set of animals was administered the drug subcutaneously (s.c.). A third set of the animals was set aside as the control and was administered normal saline either p.o. or s.c. The drug, which was previously lyophilized into a powder, was prepared by suspending the requisite quantity in

Y.R. Sohni et al./Journal o f Ethnopharmacology 45 (1995) 141-147 143

normal saline. The drug was administered p.o. with the aid of a blunt end needle and syringe. S.c. administration was through direct injection of the drug suspension with a 15 gauge needle and syringe.

2.5.1. Post-infective series. In these experiments the test extract was administered in a single dose of 15 mg, 30 mg or 60 mg to each animal in the group. The drug was administered immediately after the challenge with Salmonella typhi.

2.5.2. Pre-infective series. In these experiments, the test extract was given 24 h prior to the challenge with the bacteria. The amount of drug administered was in a single dose of 15 mg, 30 mg or 60 mg to each animal in the group.

2.5.3. Multiple dose series. The extract was given to each animal in divided doses. In one set the drug was administered prior to the challenge, in equal doses of 30 mg totalling 60 mg. In the second set 30 mg each were administered 24 h before and 24 h after the challenge with Salmonella typhi.

In the third set, 15 mg was given daily for 4 days starting 48 h prior to the infection.

In the final experiment each mouse received a total dose of 60 mg of which 30 mg was ad- ministered in doses of 15 mg each for 2 days com- mencing 48 h prior to the challenge. The remaining 30 mg were administered in doses of 10 mg each spread over a period of 72 h, after the injection of the organisms.

2.6. Controls In all experiments sets of 10-20 mice were

simultaneously inoculated with the bacteria and sham-treated with normal saline in administration schedules matching those of the test drug.

Table 1 In vitro activity of test formulation against Salmonella typhi

Test extracts Zone of inhibition MIC (ram) a (mg/ml)

Boerhavia diffusa Tinopora cordifolia 10 7

Berberis aristata 12 5 Terminalia chebula - - Zingiber officinale - - Azadirachta indica 10 8

Trichosanthes dioica Picrorrhiza kurrooa 10 8 Formulation 15 3

aZone size includes disc size which was 6 mm.

Picrorrhiza kurrooa, and the whole formulation in- hibited the growth of the test organism Salmonella typhi. The zones of inhibition ranged between 10-12 mm with MICs ranging from 5-8 mg/ml. The whole preparation demonstrated a zone of in- hibition of 15 mm and the MIC was 3 mg/ml.

The results of the in vivo tests are presented in Tables 2-4. The sham-treated control animals died within 24 h post infection. The mean survival time for 100 × LDs0 inoculum worked out to be 16.2 4- 5 h. The infection and mortality of the ani- mals was confirmed as due to Salmonella typhi by subculture of spleen, liver and blood samples. The treated mice died throughout the first 7 days post infection. Mice that survived 14 days after infec- tion were assumed to have responded to treatment. In addition, absence of Salmonella typhi was con- firmed through subculture. The results of the post- infective series are shown in Table 2. It is clear that

2. 7. Statistical analysis A simple x-square statistical analysis was per-

formed based on the survival data of control and treated animals. The P values were determined from the x-square calculations. A value of P < 0.05 was considered statistically significant.

3. Results

The results of the in vitro susceptibility tests are shown in Table 1. The extracts of Tinopora cor- difolia, Berberis aristata, Azadirachta indica,

Table 2 In vivo activity of test formulation (post-infective series)

Route of Dose Survived Percent administration (mg) mice/total survival rate

no. mice challenged

Oral

S.C.

15 0•20 0 30 0•20 0 60 0/20 0 15 0/18 0 30 0/18 0 60 4/20 20

144 E R. Sohni et al./ Journal of Ethnopharmacology 45 (1995) 141-147

Table 3 In vivo activity of test formulation (pre-infective series)

Route of Single Survived Percent administration dose mice/total survival rate

(rag) no. mice challenged

Oral

s.c.

15 0/20 0 30 4/20 20 60 4/20 20 15 0/20 0 30 4/20 20 60 8/20 40

with a single dose given orally there was no protec- tion afforded while 20% of the mice survived when the formulation was administered s.c. in a dose of 60 mg.

There was comparatively better protection observed in both the oral and s.c. sets when the drug was given prior to the challenge. Table 3 shows the results of these experiments. In the group that was administered the drug in doses of 30 mg and 60 mg p.o., 20% of the mice survived. The survival rate was 20% and 40%, respectively when 30 mg and 60 mg of the drug was injected s.c. At the low dose of 15 mg, none of the mice survived.

In the s.c. multiple-dose series, when the drug was given in doses of 30 mg each prior to infection, the survival rate was 20%. When the drug was ad- ministered in divided doses of 30 mg each before

and after the challenge, 60% of the mice survived (e < 0.025).

In the third set, the mice were administered two doses of 15 mg each before the infection with a similar dosage after the challenge and 60% of the mice survived in the s.c. set.

In the final experiment, each mouse received a total dose of 60 mg. Half of this dose was ad- ministered in 15 mg quantities 24 h and 48 h prior to the challenge with Salmonella typhi culture, and the remaining 30 mg was given in three doses of 10 mg each post-infection. When the drug was ad- ministered s.c., there was 80% survival observed (P < 0.005). In case of the group of mice treated oral- ly with the same schedule, the survival rate was 60% (P < 0.025).

4. Discussion

The test formulation has in vitro inhibitory ac- tivity against Salmonella typhi. The treatment of Salmonella typhi septicemia with the formulation appears to have a significant protective effect, even more so when administered s.c. Apart from the fact that s.c. administration induced better protec- tive effect than when given orally, a prophylactic regimen had the greatest therapeutic effect. The mice, when treated with the formulation 24/48 h prior to the challenge with Salmonella typhi and in multiple doses continuing into the post-infective phase, had a much better survival rate which was statistically significant in comparison with the sham-treated control mice. Therefore, a drug sche-

Table 4 In vivo activity of test formulation (multiple dose series)

Route of Dose (mg) administration

Day before infection Day after infection

I II I II III

Survived mice/total Percent survival no. mice challenged rate

Oral 15 15 10 10 10 s.c. 30 30

30 30 15 15 15 15 15 15 10 10 10

12/20 60 a 4/20 20

12/20 60 a 12/20 60 a 16/20 80 b

Controls: untreated mice (20/20) died within 24 h; ap < 0.025; bp < 0.005.

Y.R. Sohni et al./Journal of Ethnopharmacology 45 (1995) 141-147 145

dule that included multiple divided dose adminis- tration commencing in the pre-infective period and ending 48/72 h post-infection gave the best results.

We deliberately used a crude extract formula- tion in order to be faithful to the traditionally prescribed formulation. It would be worthwhile to study the antibacterial effect of the formulation in immunocompromised hosts particularly since the maximum therapeutic effect was observed when the formulation was given prophylactically. These studies have relevance because immunocompromi- sed patients are susceptible to infections over a long period of time. Antimicrobial prophylaxis is currently being conducted with quinolones in pa- tients with acute leukemia and recipients of bone marrow transplants (Dekker et al., 1987; Winston et al., 1987; Fu et al., 1992). Oral fluoroquinolones have been shown to be useful prophylactic an- tibacterial agents in neutropenic cancer patients with significant reductions in infections caused by members of Enterobacteriaceae (Dekker et al., 1987; Arning et al., 1990; Liang et al., 1990; Winston et al., 1990; D'Antonio et al., 1991; Kern and Kurrle, 1991). Combined drug therapy of one of the quinolones, ciprofloxacin, has been used in combination with various agents such as /3- lactams, vancomycin and others, for the treatment of febrile cases in neutropenic patients (Smith et al., 1988; Flaherty et al., 1989). However, there is now an increasing number of case reports docu- menting the development of clinical resistance to fluoroquinolones in gram-negative bacilli in- cluding Salmonella spp. (Howard et al., 1990; Pid- dock et al., 1993). The widespread prophylactic and therapeutic usage of quinolones in immuno- compromised cancer patients has raised concern about the emergence of resistance to them and a reduction in their overall impact as clinically useful antibiotics (Dholakia et a1.,1994). Since the enteric bacilli appear to be the primary target organisms of fluoroquinolone prophylaxis in im- munocompromised patients, increasing levels of resistance in these species would have a major im- pact on the efficacy of fluoroquinolone prophylax- is (Kern et al., 1994).

The formulation has displayed antibacterial ac- tivity against other intestinal pathogens such as Vibrio cholerae and Enterobacter aerogenes but not

against Escherichia coli (unpublished data). Infec- tious diseases such as typhoid continue to affect millions of people all over the world (Edelman and Levine, 1986). A major problem facing effective drug treatment of Salmonella typhi infections is the emergence of plasmid-mediated multidrug resis- tance strains. An additional difficulty with the R- factor mediated multidrug resistance is that it leads to serious complications (Madan et al., 1991; Rao et al., 1992). Multidrug treatment of cases of typhoid is not necessarily followed by a complete resolution of infection with 1-6% of all patients becoming biliary carriers and potential factors in transmission of the disease. Alternative drugs, therefore, are essential in combating the disease (Zavala-Trujillo et al., 1991). Therapy with chlor- amphenicol has little effect on the recurrence of disease relapse or chronic intestinal carriage of Salmonella typhi post therapy (Guerra et al., 1987; Gotuzzo et al., 1988). There is a need for a safe and effective oral therapy especially in case of children as the currently available oral antimicrobial thera- py is ineffective. The use of fluoroquinoline anti- microbial agents has been restricted to adults because of the risk of arthropathy during teenage years (Hooper and Wolfson, 1985; Ribard and Kahn, 1991; Naqvi et al., 1992; Bhutta et al., 1991). New chemotherapeutic drugs are needed that would be more predictably effective in areas where resistance is reported, which in turn should decrease the occurrence of post treatment typhoid relapses and chronic intestinal carriage of the organisms (Gotuzzo et al., 1994). A huge segment of the world population relies on traditional remedies to treat various diseases. Medicinal herbs and herbal products are an integral part of the tra- ditional systems of medicine (Marini-Bettolo, 1980). These are a vast emporia of potentially useful therapeutic agents. It is imperative to evalu- ate these herbs by modern scientific methods in order that these studies may lead to a better com- prehension of the phytochemical, toxicological and therapeutic aspects of these crude drugs. Such studies should also lead to novel chemotherapeutic agents.

5. Conclusion

A traditionally prescribed crude drug formula-

146 Y.R. Sohni et al./ Journal of Ethnopharmacology 45 (1995) 141-147

tion from India was evaluated for its therapeutic effect in Salmonella typhi septicemia in mice. A prophylactic dose regimen induced a significantly protective effect as compared with the untreated controls. Based on these observations further stud- ies are suggested in immunocompromised hosts. Studies on the protective efficacy of the formula- tion against other enteric pathogens would also be worthwhile.

Acknowledgement

The author Y.R.S. wishes to gratefully acknow- ledge the constant support, encouragement and liberal assistance given by Dr. P. Kale, Professor of Biology, Alabama A and M University.

References

Anderson, E.S. and Smith, H.R. (1972) Chloramphenicol resis- tance in the typhoid bacillus. British Medical Journal 2, 329-331.

Arning, M., Wolf, H.H., Aul, C., Heyll, A., Scharf. R. and Schneider, W. (1990) Infection prophylaxis in neutropenic patients with acute leukemia - a randomized comparative study with oflaxocin, ciprofloxacin, and co-trimoxazole/col- istin. Journal of Antimicrobial Chemotherapy 26 (Suppl. D), 137-142.

Barry, A.L. and Thornsberry, C. (1991) Susceptibility tests: Diffusion test procedures. In: A. Balows, W.J. Hauser Jr., K.L. Herrmann, H.D. Isenberg and H.J. Shadomy (Eds.), Manual of Clinical Microbiology, 5th edition, American So- ciety for Microbiology, Washington D.C., pp. 1117-1125.

Bhutta, Z.A., Naqvi, S.H. and Farooqui, B.J. (1991) Multidrug resistant typhoid in children; presentation and clinical fea- tures. Reviews of Infectious Diseases 13(5), 832-836.

D'Antonio, D., Iacone, A., Fioritoni, G., Betti, S., DiGirolamo, A., Piccolomini, R. and Torlontono, G. (1991) Antibac- terial prophylaxis in granulocytopenic patients: a rando- mized study of oflaxocin versus norfloxacin. Current Therapeutic Research 50, 304-311.

Dekker, A.W., Rozenberg-Arska, M. and Verhoef, J. (1987) In- fection prophylaxis in acute leukemia: a comparison of ciprofloxacin with trimethoprim-suifamethoxazole and col- istin. Annals of Internal Medicine 106, 7-12.

Dholakia, N., Rolston, K.V.I., Ho, D.H., LeBlanc, B. and Bodey, G.P. (1994) Susceptibilities of bacterial isolates from patients with cancer to levofloxacin and other quinolones. Antimicrobial Agents and Chemotherapy 38(4), 848-852.

DuPont, H.L. (1993) Quinolones in Salmonella typhi infection. Drugs 45, (Suppl. 3), 119-124.

Edelman, R. and Levine, M.M. (1986) Summary of an interna- tional workshop on typhoid fever. Reviews of Infectious Diseases 8, 329-349.

Eykyn, S.J. and Williams, H. (1987) Treatment of multiresis- tant Salmonella typhi with oral ciprofloxacin. Lancet 2, 1407-1408.

Flaherty, J.P., Waitley, D., Edlin, B., George, D., Arnow, P., O'Keefe, P. and Weinstein, R.A. (1989) Multicenter, rando- mized trial of ciprofloxacin plus azlocillin versus ceftazidine plus amikacin for empiric treatment of febrile neutropenic patients. American Journal of Medicine 87, (Suppl. 5A), 2783-2825.

Fu, K.P., Lafredo, S.C., Foleno, B., Isaacson, D.M., Barrett, J.F., Tobia, A.J. and Rosenthale, M.E. (1992) In vitro and in vivo bacterial activities of levofloxacin (L-oflaxocin), an optically active oflaxocin. Antimicrobial Agents and Chemo- therapy 36, 860-866.

Goldstein, F.W., Chumpitaz, J.C., Guevara, J.M., Papado- poulous, B. and Acar, J.F. (1986) Plasmid-mediated resis- tance to multiple antibiotics in Salmonella typhi. Journal of Infectious Diseases 153, 261-266.

Gotuzzo, E., Benavente, L., Guerra, J., Maguina, C. and Car- rillo, C. (1988) Relapse in typhoid fever. Proceedings of the International Congress for Infectious Diseases, Rio De Janeiro, Brazil. International Society for Infectious Diseases, abstract 496, p 44.

Gotuzzo, E., Echevarria, J., Carrillo, C., Sanchez, J., Grados, P., Maguina, C. and DuPont, H.L. (1994) Randomized comparison of aztreonam and chloramphenicol in treat- ment of typhoid fever. Antimicrobial Agents and Chemother- apy 38(3), 558-562.

Guerra, J., Benavente, L., Gotuzzo, E., Grados, O. and Bravo, N. (1987) Predictive value of stool culture and bile culture post-treatment of typhoid fever (TF) in the identification of relapse and chronic cartier state (CC), Program Abstracts 27th Interseience Conference on Antimicrobial Agents and Chemotherapy, American Society for Microbiology, Washington, DC., abstract 1169.

Hooper, D.C. and Wolfson, J.S. (1985) The fluoroquinolones: pharmacology, clinical uses and toxicities in humans. Anti- microbial Agents and Chemotherapy 28, 716-721.

Howard, A.J., Joseph, T.D., Bloodworth, L.L.O., Frost, J.A., Chart, H. and Rowe, B. (1990) The emergence ofciproflox- acin resistance in Salmonella typhimurium. Journal of Anti- microbial Chemotherapy 26, 196-198.

Kern, W.V. and Kurrle, E. (1991) Oflaxocin versus trime- thoprim-sulfamethoxazole for prevention of infection in pa- tients with acute leukemia and granulocytopenia. Infection 19, 73-80.

Kern, W.V., Andriof, E., Oethinger, M., Kern, P., Hacker, J. and Marre, R. (1994) Emergence of fluoroquinolone- resistant Escherichia coli at a cancer center. Antimicrobial Agents and Chemotherapy 38(4), 681-687.

Liang, R.H.S., Yung, R.W.H., Chan, T.K., Chau, P.Y., Lam, W.K., So, S.Y. and Todd, D. (1990) Oflaxocin versus co- trimoxazole for prevention of infection in neutropenic pa- tients following cytotoxic chemotherapy. Antimicrobial Agents and Chemotherapy 34, 215-218.

Madan, A., Dhar, A., Kulshreshtha, P.D., Laghate, V.D. and Dhar, D. (1991) Preliminary observation on drug resistant cases of typhoid fever. Journal of the Association of Physi- cians of India 39(6), 449-451.

Y.R. Sohni et al./ Journal of Ethnopharmacology 45 (1995) 141-147 147

Marini-Bettolo, G.B. (1980) Present aspects of the use of plants in traditional medicine. Journal of Ethnopharmacology 2, 5-7.

Naqvi, S.H., Bhutta, Z.A. and Farrooqui, B.J. (1992) Therapy of multidrug resistant typhoid in 58 children. Scandinavian Journal of Infectious Diseases, 24(2), 175-179.

Parashar, A.S. (1961) Sharangdhar Samhita, Shri Baidyanath Ayurved Bhavan, Calcutta, India p 211.

Piddock, L.J.V., Griggs, D.J., Hall, M,C. and Jin, Y.F. (1993) Ciprofloxacin resistance in clinical isolates of Salmonella typhimurium obtained from two patients. Antimicrobial Agents and Chemotherapy 37, 662-666.

Rao, R.S., Amarnath, Sujatha, S. (1992) An outbreak of typhoid due to multidrug resistant Salmonella typhi in Pon- dicherry. Transactions of the Royal Society for Tropical Medicine and Hygiene 86(2), 204-205.

Reed, L.J. and Muench, H. (1938) A simple method of estimating fifty percent end points. American Journal of Hygiene 27, 493-497.

Ribard, P. and Kahn, M.F. (1991) Rheumatological side- effects of quinolones. In: M.F. Kahn (Ed.), Bailliere's Clini- cal Rheumatology; drug-induced rheumatic diseases. Bailliere Tindall, London, pp. 15-191.

Sahm, D.F. and Washington, J.A. II (1991) Antibacterial sus-

ceptibility tests: Dilution methods. In: A. Balows, W.J. Hauser Jr., K.L. Herrmann, H.D. Isenberg and H.J. Shadomy (Eds.), Manual of Clinical Microbiology, 5th edi- tion. American Society for Microbiology, Washington, D.C., pp. 1105-1116.

Smith, G.M., Leyland, M.J., Farrel, I.D. and Geddes, A.M. (1988) A clinical, microbiological, and pharmacokinetic study of ciprofloxacin plus vancomycin as initial therapy of febrile episodes in neutropenic patients. Journal of Anti- microbial Chemotherapy 21,647-655.

Winston, D.J., Ho, W.G., Champlin, R.E., Karp, J., Bartlett, J., Finley, R.S., Joshi, J.H., Talbot, G., Levitt, L., Deresin- ski, S. and Corrado, M. (1987) Norfloxacin for prevention of bacterial infections in granulocytopenic patients. Ameri- can Journal of Medicine 82, (Suppl. 6B), 40-46.

Winston, D.J., Ho, W.G., Bruckner, D.A., Gale, R.P. and Champlin, R.E. (1990) Oflaxocin versus vancomycin/poly- myxin for prevention of infections in granulocytopenic pa- tients. American Journal of Medicine 88, 36-42.

Zavala-Trujillo, I., Quiroz, C., Gutierrez, MA., Arias, J. and Rentaria, M. (1991) Fluoroquinolones in the treatment of typhoid fever and the carrier state. European Journal of Clinical Microbiology and Infectious Diseases 10(4), 334-341.