Bull. Org. mond. Sante 1967, 37, 763-771 Bull. Wid Hlth Org.

Drug-sensitivity of El Tor Vibrio Strains Isolated in the Philippines in 1964 and 1965*

SHOGO KUWAHARA, SACHIKO GOTO, MASATAKE KIMURA & HISAO ABE

About 1500 strains ofEl Tor vibrios, isolated in 1964 and 1965 in the Philippines, were examinedfor their susceptibilities to 17 drugs. All the strains tested were highly sensitive to dihydroxymethyl-furalazine, and most were highly sensitive to tetracycline hydrochloride, chloramphenicol and erythromycin, and moderately sensitive to novobiocin, dihydro- streptomycin sulfate, kanamycin and neomycin. They showed a remarkable fluctuation of sensitivity to ampicillin, cefaloridine, cefalotin and sulfafurazole, and a high resistance to benzylpenicillin sodium, oleandomycin and spiramycin. Experimental confirmation wasprovided ofthefact that El Tor vibrios and non-agglutin- able vibrios can be distinguished from classical cholera vibrios by their resistance to poly- myxin B and colistin. Highly streptomycin-resistant strains, and to a lesser extent ampicillin- and sulfafura- zole-resistant strains, were relatively often isolated from cholera patients who had been treated with antibiotics. One patient yielded a strain resistant to tetracycline, chloram- phenicol, streptomycin and sulfafurazole.

Since the discovery of broad-spectrum antibiotics Ninety-four strains in total were used as controls, and antibiotics effective against Gram-negative including El Tor vibrios isolated in India in 1964 bacteria, the sensitivities of strains of Vibrio cholerae and 1965, classical cholera vibrios, and non-agglutin- to these drugs have been studied by Felsenfeld et al. able (NAG) vibrios isolated since 1961. (1951), Felsenfeld & Soman (1952), Pollitzer (1959) and others. Tetracycline derivatives, chloram- Drugs tested phenicol and streptomycin and related compounds were confirmed to be highly effective in vitro. Fifteen antibiotics and 2 synthetic chemothera- However, the only study known to the authors on peutics were used: tetracycline hydrochloride, the sensitivity pattern of El Tor vibrios, the prevalent chloramphenicol, dihydrostreptomycin sulfate, kana- strain in south-eastern Asia, is that by Kao (1962) on mycin sulfate, erythromycin, ampicilin, benzyl- the strains isolated in Taiwan. The present report penicillin sodium, oleandomycin, spiramycin, neo- deals chiefly with the sensitivity pattern of about mycin, colistin (both as the sulfate and as the 1500 fresh strains of El Tor vibrios which were methanesulfonate), polymyxin B, cefaloridine, cefa- isolated in the Philippines in 1964 and 1965. lotin, novobiocin, sulfafurazole and dihydroxy- methyl-furalazine. MATERIALS AND METHODS Strains used Determination ofsensitivity Out of the vibrio strains isolated in the Philippines Sensitivity was determined by the agar-plate in 1964 and 1965 and brought to Japan, 1550 strains dilution method advocated by Fukumi, using heart- identified as El Tor vibrios were used in this experi- infusion agar: the drugs were mixed in different con- ment; these strains were divided into 6 groups centrations with the melted agar (at about 50°C) according to the time and place of isolation, as and then poured into plates, which were streaked with listed in Table 1. the overnight broth cultures of the organisms in the marked zones. Mueller-Hinton agar was used for * From the Department of Health, Japan. the sulfafurazole-sensitivity.

2107 763 764 S. KUWAHARA AND OTHERS

TABLE I GROUPING OF TESTED STRAINS

Group No. of strains Place of isolation Date of isolation.

El Tor vibrios 169 Davao, Philippines 1964 11 384 Bacolod, Philippines May-August 1964 III 96 Manila, Philippines 1964 IV 499 Bacolod, Philippines 1964 V 402 Unspecified, Philippines 1964-65 VI 12 India Unspecified

V. cholerae 42 Unspecified 1961-64 NAG vibrios 40 Unspecified 1961-64

RESULTS Considerable fluctuation occurred in both strepto- mycin- and kanamycin-sensitivities according to the Drug-sensitivity patterns place from which the strain was isolated. Sensitivities to ampicillin varied over a relatively The sensitivity of the majority of the El Tor wide range; strains resistant to above 100 ,ug/ml vibrios to tetracycline, chloramphenicol, strepto- accounted for 6.5 % of the total. mycin, kanamycin, erythromycin, ampicillin, sulfa- The sulfafurazole-sensitivity also showed remark- furazole and furalazine was determined, and some able fluctuations depending on the place of isolation; strains were examined for their sensitivity to other resistant strains were especially numerous in the antibiotics. The results are shown in Tables 2 and 3. Davao group (Group I). Furalazine showed the highest vibriostatic activity: The sensitivities of each strain to antibiotics other the growth of 79 % of the strains tested was inhibited than the 8 above-mentioned ones were also de- by concentrations of less than 0.19 ,ug/ml. Tetra- termined. As for the antibiotics of the macrolide cycline was also very active, inhibiting the growth of group other than erythromycin, it was found that 81 % of the strains tested at concentrations of spiramycin and oleandomycin only showed bacterio- 3.12 ,ug/ml or less. Chloramphenicol also inhibited static activity at concentrations above 100 ,tg/ml. the growth of the majority of the strains at concentra- Benzylpenicillin sodium manifested less spread tions ranging from 0.78 ,tg/ml to 3.12,g/ml, although in antibacterial activity than ampicillin. It is note- strains resistant to 25 ,tg/ml or more were detected worthy that the former inhibited the growth of all more frequently with chloramphenicol than with the strains tested at 100 jug/ml, while some strains tetracycline. were resistant to the latter at this level. Neomycin Erythromycin also exhibited high antibacterial showed roughly the same activity as benzylpenicillin activity, inhibiting the growth of more than 90 % of sodium. the tested strains at a concentration of 12.5 jug/ml or The strains tested showed a relatively high sen- less. sitivity to novobiocin; 90% were inhibited at con- Many of the test strains were less sensitive to centrations of 6.25 ,ug/ml or lower. streptomycin and kanamycin than the above 4 anti- As for the cefalosporin derivatives, the suscep- biotics; the minimum inhibitory concentrations tibility of the El Tor strains to cefalotin was some- ranged from 6.25 ,ug/ml to 50 ,g/ml for the majority what higher than that to cefaloridine: 30 out of of the strains. The over-all kanamycin-sensitivity 38 strains tested were inhibited by 6.25 ,ug/ml- was a little higher than that to streptomycin: there 3.12 jug/ml of cefalotin, whereas the minimum in- were some highly streptomycin-resistant strains hibitory concentration (MIC) of cefaloridine was withstanding 200 ,g/ml of this drug, whereas none 12.5 jtg/ml or more, and 6 out of 38 strains were were resistant to above 50 jtg/ml of kanamycin. resistant to 100 ,tg/ml of the latter drug. DRUG-SENSITIVITY OF EL TOR VIBRIO STRAINS IN THE PHILIPPINES 765

TABLE 2 DISTRIBUTION OF DRUG-SENSITIVITY OF TESTED STRAINS I No. of strains inhibited Drug Group strains Minimal inhibitory concentration (Mg/ml) <0.19 )0.39 0.78 1.56 3.12 6.25 12.5 25 50 100 >100 200 >200 I I I I I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Dihydroxy- methyl- ,II, IV-VI 1 440 a 1 132 233 51 22 2 furalazine

166 1 87 74 3 II 381 86 275 19 I III 96 3 89 4 IV 499 373 120 5 Tetra- V 402 402 cycline hydro- VI 12 12 chloride Total 1 556 1374 222 1570 7 87 275 20 1

V. cholerae 1 42 42 NAG 40 39

167 5 16 100 43 2 II 381 5 9 237 129 I III 96 14 76 2 2 IV 499 18 10 337 129 2 V 402 9 393 Chloram- phenicol VI 12 12

Total 1 557 24 45 534 420 526 3 1 2

V. cholerae 42 5 3 34 NAG 40 39

168 2 11 31 35 68 17 4 II 363 2 6 12 6 23 314 III 96 15 77 4 IV 499 2 135 313 47 2 Erythro- V 402 3 397 1 1 mycin VI 12 12

Total 1 540 4 17 43 43 253 724 452 2 1 I

V. cholera 42 11 24 3 2 NAG 40 14 16 10

a Only pooled data are available for this drug. 766 S. KUWAHARA AND OTHERS

TABLE 2 (concluded) No. of strains inhibited Drug Group sNtroanof Minimal inhibitory concentration (j.g/ml) <0.191 0.39 0.78 1.56 3.12 6.25 12.5 25 50 100 >100 200 >200

169 107 6 49 7 II 373 337 I 35 Ill 96 3 87 4 2 IV 499 I 10 108 343 15 3 19 Dihydro- V 402 19 366 4 13 strepto- mycin VI 12 8 3 sulfate Total 1 551 107 6 1 14 608 723 20 3 69

V. cholerae 42 1 12 27 2 NAG 40 24 14

169 5 31 77 56 II 362 I 1 3 15 305 36 Ill 96 2 75 69 IV 499 2 27 351 119 lV 402 402 Kanamycin VI 12 12

Total 1 540 6 1 1 3 1 5 340 215 438 521 ,~ ,. ,~ ,- ,-~-. l~ V. cholerae 42 1 2 17 22 NAG 40 10 29 ,- -, 167 8 63 48 20 23

II 373 16 29 37 61 80 86 22 42

Ill 96 4 27 17 4 6 37 IV 398 27 84 141 83 29 19 15 V 402 402 Ampicillin VI 12 5 7

Total 1 448 24 120 173 254 612 119 47 ] 94

V. cholerae 42 8 34 NAG 40 4 4 4 21 1 6

Minimal inhibitory concentration (Mg/ml) 10 100 1000 >1 000

168 11 9 148 II 373 349 23 1 II 92 65 6 21 IV 499 282 193 9 15 Sulfafur- V 402 13 367 18 4 azole VI 12 12 Total 1 546 709 600 48 189

V. cholerae 42 22 20 NAG 40 2 23 15 DRUG-SENSITIVITY OF EL TOR VIBRIO STRAINS IN THE PHILIPPINES 767

TABLE 3. DISTRIBUTION OF DRUG-SENSITIVITY OF TESTED STRAINS No. of strains inhibited No. of Drug Group strains Minimal inhibitory concentration ("tg/ml) <0.19 0.39 0.78 1.56 3.12 6.25 12.5 25 50 100 >100

Neomycin IV 99 1 9 4 85 Benzylpenicillin sodium IV 99 3 9 52 32 3 Novobiocin VI 97 2 1 26 25 36 7 Cefaloridine II 38 6 20 6 6 Cefalotin II 38 20 10 4 1 3 Spiramycin III 96 96 Oleandomycin III 96 96

The drug-sensitivity of V. cholerae control strains being as high as 500 units/ml for polymyxin B and to the last-mentioned 7 antimicrobial agents tended above 1000 units/ml for the colistin derivatives. to be higher than that of El Tor vibrios. It was not On the other hand, all strains of V. cholerae were known, however, whether this was due to the age inhibited by less than 100 units/ml of polymyxin B of the V. cholerae cultures used in this experiment. and below 500 units/ml of the colistin derivatives-a NAG vibrios showed nearly the same degree of striking difference in sensitivity. sensitivity as El Tor vibrios. Comparison of sensitivity patterns of strains from Sensitivities to polymyxin-B and colistin derivatives antibiotic-treated and untreated patients The sensitivities of El Tor vibrios, V. cholerae Strains isolated from El Tor cholera patients and NAG vibrios to polymyxin B, colistin sulfate treated with tetracycline, chloramphenicol, kana- and colistin methanesulfonate are summarized in mycin, erythromycin and furalazine were compared Table 4. with those from untreated cases with respect to their Both El Tor and NAG vibrios were highly re- sensitivity to these antibiotics. The results are shown sistant to both polymyxin B and colistin, the MIC in Table 5.

TABLE 4. SENSITIVITY OF VIBRIOS TO COLISTIN AND POLYMYXIN B No. of strains inhibited Drug Organism Group No. of Minimal inhibitory concentration (units/ml) a 2.5 25 50 100 250 500 1000 >1000 5000 >5000

Colistin El Tor IV-VI 812 I 812 sulfate V. cholerae 42 9 21 12 NAG 40 40

Colistin El Tor IV-VI 812 10 802 methane- sulfonate V. cholerae 42 15 14 13 NAG 40 40

Polymyxin El Tor IV-V 800 2 4 794 B V. cholerae 42 13 22 6 1 NAG 40 5 8 6 21

a 1mg of polymyxin B = 7100 units; 1 mg of colistin sulfate= 18500 units; 1 mg of colistin methanesulfonate= 12500 units. 768 S. KUWAHARA AND OTHERS

TABLE 5 DISTRIBUTION OF SENSITIVITY OF STRAINS ISOLATED FROM DRUG-TREATED AND UNTREATED PATIENTS (GROUP 1I1)

No. of strains inhibited

screened ment a strains Minimal inhibitory concentration (pg/ml) <0.19 0.39 0.78 |1.56 3.12 6.25 1112.5 25 50 100 >100 200 >200 _~~~ 1=

None 15 3 12 TC 16 15 Tetracycline CM 10 9 hydrochloride PS 5 5 EM 6 6 KM 44 42 2 1~ I~I~1-1~

None 15 9 1 6 1 1 TC 16 5 9 Chloramphe- CM 10 10 nicol PS 5 5 EM 6 5 1 KM 44 1 41 2

None 15 2 13 TC 16 15 I Dihydro- streptomycin CM 10 10 sulphate PS 5 5 EM 6 5 KM 44 39 3 2

None 15 15 TC 16 16 CM 10 10 Kanamyci n PS 5 4 EM 6 4 KM 44 26 18

None 15 5 10 TC 16 4 9 3 CM 10 9 1 Erythromycin PS 5 5 EM 6 5 KM 44 5 39

None 15 2 11 2 TC 16 2 5 4 5 CM 10 2 6 Ampicillin PS 5 1 4 EM 6 1 1 1 2 KM 44 8 4 2 5 25

MIC (Mg/ml) 1.9 3.9 7.8 15.6 31.2 62.5 125 250 500 >1 000

None 15 8 3 2 TC 16 6 7 2 CM 10 1 1 7 1 Sulfafurazole PS 5 Not tested 1 3 EM 6 2 2 KM 44 3 8 13 2 1 16

a TC= Tetracycline hydrochloride; CM = chloramphenicol; PS = dihydroxymethyl-furalazine; EM = erythromycin; KM = kanamycin. DRUG-SENSITIVITY OF EL TOR VIBRIO STRAINS IN THE PHILIPPINES 769

Highly streptomycin-resistant strains were isolated TABLE 6 from the kanamycin-treated cases, indicating a close COMPARISON OF SENSITIVITIES OF FIRST AND LAST cross-reaction between these 2 drugs. With respect ISOLATES FROM THE SAME PATIENT a to the other drugs, no difference was observed be- Minimal inhibitory Interval tween the strains isolated from the treated and Case Drug b concentration (gg/ml) between untreated cases. ___ ,~ First isolate Last isolate (days) Considerable fluctuations were observed in ampi- cillin- and sulfafurazole-sensitivity among the strains I SM 25 >200 5 isolated from the treated patients; the cause of these 2 SM > 200 > 200 6 fluctuations is unknown. 3 SM >200 >200 2 Sensitivities ofstrains isolatedfrom the same cases at 4 SM - > 200 - different stages of illness 5 SM 25 > 200 6 Some of the group-II strains were isolated twice. 6 SM 25 >200 1 Differences in sensitivity patterns were observed 7 SM 25 > 200 3 between the 2 isolates in 28 instances out of the 114 8 SM 25 > 200 4 group-IT strains studied; see Table 6. 9 SM 25 > 200 4 The first isolates in 17 of these 28 cases were streptomycin-sensitive, whereas the last isolates 10 SM > 200 > 200 2 were highly streptomycin-resistant, and vice versa 11 SM >200 >200 2 in 10 cases. In one case a multiple-resistant strain 12 SM 25 >200 7 (tetracycline, chloramphenicol, streptomycin and 13 SM 25 >200 7 sulfafurazole) was detected in the last isolation. 14 SM 25 > 200 3 15 SM 25 >200 3 DISCUSSION 16 SM 25 >200 3 Felsenfeld et al. (1951) and Chaudhuri et al. (1950) 17 SM 25 >200 4 confirmed that oxytetracycline and chlortetracycline 18 SM >200 >200 7 are effective against cholera vibrios. The present study demonstrated that not only tetracycline but 19 SM >200 >200 3 also chloramphenicol, erythromycin and in parti- 20 SM >200 >200 1 cular furalazine were highly active and streptomycin, 21 SM >200 25 1 kanamycin and novobiocin moderately active 22 SM >200 >200 2 against both classical and El Tor vibrios in vitro. 23 SM 25 > 200 2 On the other hand, the vibriostatic activities of spiramycin, oleandomycin and benzylpenicillin 24 SM 25 > 200 4 sodium were extremely low, and those of cefalori- 25 SM >200 25 3 dine, cefalotin, ampicillin and sulfafurazole under- 26 SM 25 > 200 4 went considerable fluctuation. 27 SM 25 > 200 4 The results of the clinical trials on the effectiveness 28 TC 6.25 100 of antibiotics against El Tor cholera in the Philip- pines (Kobari, Uylangco & Vasco, 1967; Uylangco 28 CM 1.56 100 et al., 1967; Kobari, Uylangco et al., 1967) 1 are in ac- 28 SM 25 >200 cordance with those of the present in vitro experi- 28 SA 3.9 >1000 ments. Although the non-absorbable antibiotics, a Resistant strains were obtained from 28 out of 114 cases such as streptomycin and kanamycin, manifested a examined. relatively low antibacterial activity in the present b SM= dihydrostreptomycin sulfate; TC= tetracycline hydro- experiments, it could not be determined whether their chloride; CM= chloramphenicol; SA= sulfafurazole. low clinical effectiveness was due to their low vibriostatic activity or to their non-absorbable The high vibriostatic activity of polymyxin B character. against classical cholera vibrios has been pointed out by Haldar et al. (1961). Later, Gan & Tjia (1963) 1 See the papers on pp. 751, 810 and 812 of this issue. and Powell & Finkelstein (1964) showed that El Tor 770 S. KUWAHARA AND OTHERS vibrios were highly resistant to polymyxin B, and mycin and kanamycin, was observed in 28 out of that this could be used as a method for the differen- the 114 cases studied. The report by Gohar et al. tiation of these two groups. Abe et al. (1966) (1953) that cholera vibrios easily acquired a high observed a similar difference in sensitivities to streptomycin-resistance would support the finding colistin sulfate and colistin methanesulfonate be- of highly streptomycin-resistant strains in the pre- tween classical cholera and El Tor vibrios, and sent experiments. stated that these two groups can be distinguished Unpublished experiments by us showed that a from each other with a disc containing 50 units- multiple-antibiotic-resistant strain could transmit its 100 units of polymyxin B, 150 units ofcolistin sulfate resistance through conjugation to sensitive strains of or 1000 units of colistin methanesulfonate. Our Enterobacteriaceae such as Salmonella and Shigella, results confirm this in general. thus confirming with natural strains the possibility Differences in antibiotic-sensitivities between anti- of transmission of the R factor between cholera biotic-treated and untreated patients were observed vibrios and Enterobacteriaceae, demonstrated ex- only with regard to kanamycin treatment. The perimentally by Kuwahara et al. (1963). fluctuation of sensitivity to ampicillin and sulfa- Felsenfeld et al. (1951) pointed out the increase in furazole found among strains from the antibiotic- drug-sensitivity of cholera vibrios which occurs treated patients cannot be explained causatively during successive subcultures. It is well known that within the range of the present studies because of the growth and enzyme activities of cholera and El lack of knowledge of the details of treatments of Tor vibrios fall after successive transfers on nutrient investigated cases. media. It is not known whether the strains used in A remarkable difference in sensitivity between 2 the present experiments retained their original sus- isolates from the same patient, limited to strepto- ceptibilities throughout the study.

RESUME

Les types de sensibilite aux m6dicaments de 1550 taines souches se sont montrees resistantes a 200 /Ag/ml souches de vibrions El Tor isol6es aux Philippines en de streptomycine. 1964 et 1965 ont 6te etudies au Japon. Des souches de On a observ6 que la sensibilite a l'ampicilline variait vibrions El Tor isolees en Inde, de vibrions choleriques dans des limites assez larges, et 6,5 % des souches etu- classiques et de vibrions non agglutinables (NAG) ont diees ont resiste i 100 ,ug/ml. La gamme de sensibilit6 servi de temoins. On a d6termine la sensibilit6 par la ai la benzylpenicilline sodique etait plus etroite. Dans le m6thode de dilution sur plaques de g6lose, en utilisant groupe des cefalosporines, la cefalotine se montrait lege- une gelose 'a l'infusion de cceur, sauf dans le cas du rement plus active que la cefaloridine, la concentration sulfafurazol pour lequel on a utilise la gelose Mueller- minimale active de ces deux medicaments variant pour Hinton. le premier de 6,25 Lg/ml 'a 3,12 &g/ml et depassant pour La dihydroxymethyle-furalazine s'est revel6e le plus le second 12,5 tg/ml. La sensibilite au sulfafurazol etait actif des medicaments mis 'a l'epreuve: elle a supprime faible dans l'ensemble et variait de facon remarquable 80 % environ des souches &laconcentration de0, 19 g/ml. en fonction du district oui l'on avait isol6 les vibrions. La t6tracycline et le chloramphenicol ont aussi fait L'ol6andomycine et la spiramycine n'ont pu empecher preuve d'une forte activite vibriostatique; environ 50 % la croissance d'aucune des souches eprouv6es tant que des souches ont ete supprimees par la tetracycline a des leur concentration n'a pas atteint 100 tg/ml. concentrations s'etageant entre 0,78 sg/ml et 3,12 ug/ml, En general, les vibrions NAG ont fait preuve d'un tandis que le chloramphenicol, dans les memes limites degre de sensibilit6 presque identique a celui des vibrions de concentration, a inhibe la croissance de la majorite El Tor, alors que les souches de cholera classiques des souches d'epreuve. L'erythromycine a ete efficace etaient l6gerement plus sensibles. I1 a 6t6 confirme que vis-a-vis de la plupart des souches a une concentration les souches de vibrions El Tor et NAG etaient tres egale ou inferieure i 12,5 .g/ml, alors que la novobiocine resistantes 'a la colistine et a la polymyxine B, tandis a aussi fait preuve d'une activite appreciable, inhibant que les souches classiques de Vibrio cholerae etaient, au la croissance de nombreuses souches a une concentra- contraire, tres sensibles ai ces deux antibiotiques. tion egale ou inferieure a 6,25 ,g/ml. Un grand nombre On a isole des souches tres resistantes a la strepto- de souches ont et moins sensibles a la streptomycine, mycine chez des malades atteints de cholera El Tor et ai la neomycine et a la kanamycine, la concentration traites Li la kanamycine, ce qui indique une forte r6sis- inhibitrice minimale allant de 6,25 jug/ml a 50 ,g/ml; cer- tance croisee envers ces deux medicaments. Dans les DRUG-SENSITIVITY OF EL TOR VIBRIO STRAINS IN THE PHILIPPINES 771 cas de traitement par la tetracycline, le chloramphenicol chez les memes malades ont fait preuve de types de ou 1'erythromycine, on n'a observe aucune difference sensibilite differents. Chez un malade, on a isole, dans entre les souches isol6es dans les groupes traites et les dernires selles, une souche tr6s resistante au sulfa- non traites. furazol, A la streptomycine, au chloramphenicol et a la Dans 28 cas, les premiers et les demiers vibrions isoles tetracycline.

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