Vol. 18, No. 1ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, JUly 1980, p. 182-1890066-4804/80/00-0182/08$02.00/0

Comparative Bactericidal Effects of Azlocillin and TicarcillinAgainst Pseudomonas aeruginosaA. R. WHITE, K. R. COMBER, AND R. SUTHERLAND*

Beecham Pharmaceuticals Research Division, Brockham Park, Betchworth, Surrey, England

Azlocillin was relatively ineffective against actively growing cultures of Pseu-domonas aeruginosa in tests of bacteriolytic and bactericidal activity in whichticarcillin demonstrated pronounced bactericidal effects over a wide range ofconcentrations. Microscopic observation showed that azlocillin generally inducedthe formation of filamentous cells of P. aeruginosa which lysed only slowly, butticarcillin caused the production of spheroplasts and subsequent rapid lysis.During the course of the bactericidal tests, azlocillin was inactivated, presumablyby the fi-lactamase produced by P. aeruginosa, and the filamentous cells resumednormal cell division and growth. In contrast, there was no loss of ticarcillinactivity, and there was no evidence of resumption of growth of P. aeruginosa inthe presence of ticarcillin. These results suggest that the different bactericidaleffects demonstrated by azlocillin and ticarcillin against P. aeruginosa are relatedprimarily to dose-related differences in inhibition of cell wall synthesis andsecondarily to the instability of azlocillin to pseudomonal /3-lactamase.

There are at present two classes of penicillinsknown to possess significant activity againstPseudomonas aeruginosa. These comprise thea-carboxypenicillins, carbenicillin and ticarcillin,possessing an acidic group in the side chain ofthe molecule, and the acylaminopenicillins, de-rivatives of ampicillin or amoxycillin with a basicgroup in the side chain (12). Examples of thelatter group include the two ureidopenicillins,azlocillin and mezlocillin (3, 7, 8, 15), which areavailable in Europe, and apalcillin (4, 10, 11) andpiperacilin. (6, 14, 17, 18), which are in wide-spread clinical trial.From the results of published studies it is

evident that there are pronounced differencesbetween the a-carboxypenicillins and the ac-ylaminopenicillins with regard to the antibacte-rial effects produced against P. aeruginosa. Forinstance, the antipseudomonal activity of thelatter is greatly influenced by inoculum size (3,6, 15), and this has been correlated with insta-bility to the f8-lactamase of P. aeruginosa (2).In addition, the bactericidal activity of ureido-penicillins against P. aeruginosa has been re-ported to be much less than that of carbenicillinor ticarcillin (6, 7, 13, 14), although in otherstudies little or no difference was observed inthis respect between the two classes of penicil-lins (5, 15, 18).

This study was designed to compare the bac-tericidal effects produced by azlocillin and ticar-cillin against P. aeruginosa, as measured bytests of bacteriolytic activity, viable count stud-ies, and observation of the morphological effectsproduced by the two penicillins.

MATERIALS AND METHODS

Antibiotics. Azlocillin (sodium) was a laboratorypreparation provided by Bayer AG (Leverkusen, Fed-eral Republic of Germany), and ticarcillin (disodium)was laboratory reference standard material (BeechamPharmaceuticals, Worthing, England).

Cultures. P. aeruginosa ATCC 27853 and P.aeruginosa NCTC 10662 are standard laboratorystrains employed for antibiotic susceptibility testing(9, 16). The strain of P. aerugmosa 11 is a clinicalisolate that has been used in these laboratories forchemotherapeutic studies.MICs. The minimum concentrations of azlocillin

and ticarcillin required to inhibit growth of the teststrains of P. aeruginosa (minimal inhibitory concen-tration, MIC) were determined by serial dilution in0.05-mi volumes ofMueller-Hinton broth (Difco) usingthe microtiter technique (1). The inoculum, 0.05 ml ofan overnight broth culture, was diluted to give finalcell densities of about 108 cells/ml, and 106 cells/mlwas added to the wells with a pipette. The MICs weremeasured after incubation at 37°C for 18 h.

Bacteriolytic effects. Prelimninary tests were per-formed in a Bonet-Maury continuous recording bio-photometer (Jobin-Yvon, Long Jumeau, France), butclumping of the organisms caused unsatisfactory re-cording of opacity. Accordingly, the strains of P.aeruginosa were grown in shaken culture, and theoptical density of samples taken at intervals duringthe test was measured in an EEL nephelometer (EvansElectroselenium Ltd., Halstead, England) as follows.Erlenmeyer flasks (100 ml) containing 18 ml of double-strength veal infusion (Difco) were inoculated with 0.1ml of a 1:1,000 dilution of a 5-h shake culture of thetest organism. The flasks were incubated in an orbitalshaker (G4, New Brunswick Scientific Co.) for 4 h at37°C, at which time prewarmed veal infusion contain-ing the antibiotics was added to the flasks to give the

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requisite final concentrations. At this point, the loga-rithmic-phase cultures contained approximately 2 x108 cells/ml and produced an optical density readingof 20 to 30 U in the EEL nephelometer using an OR2red filter. The optical density of the shake cultureswas measured at frequent intervals for a period of 4 h.

Viable count tests. The antibiotics were added toshake cultures of the test strains of P. aeruginosaprepared as above. Samples were removed at intervalsand diluted in double-strength veal infusion, and 0.02-ml volumes were pipetted onto nutrient agar platescontaining penicillinase (Difco). Colony counts wereperformed after overnight incubation of the plates at37°C. In certain tests, the concentrations of penicillinspresent in the cultures of P. aeruginosa were mea-sured by agar-diffusion microbiological assay using P.aeruginosa NCTC 10701 as assay organism.

Morphological effects. Samples were taken fromthe shake cultures during the course of the bactericidaltests described above and carefully mixed with equalvolumes of 2.0% (wt/vol) glutaraldehyde fixative indouble-strength veal infusion. The osmolality of dou-ble-strength veal infusion (253 mOs/kg) was similar tothat of pooled human serum (270 mOs/kg). The glu-taraldehyde fixative was shown in preliminary exper-iments to preserve the morphology of the bacteria andto prevent any further antibacterial activity of thepencillins. Specimens for microscopy were mountedon a thin film of agar prepared by spreading 0.8% agar(Difco) over the surface of a glass cover slip (22 by 50mm) which was trimmed to a 10-mm square after theagar had solidified. The sample was placed in thecenter of the agar and covered with a second cover slipfor high-resolution microscopy using a Zeiss WL mi-croscope fitted with differential interference contrastoptics.

RESULTSMICs. The results in Table 1 show the MICs

of azlocillin and ticarcillin against the three teststrains of P. aeruginosa. The activity of azlocil-lin was greatly influenced by the size of inocu-lum, and the compound was relatively inactivewhen tested against 108 cells/ml, but showed ahigh level of activity against 106 cells/ml (MIC= 4.0 jig/ml). The inoculum effect observed withticarcillin was much less than that obtained withazlocillin, and ticarcillin was more active thanazlocillin against the larger inoculum, whereasazlocillin was the more active of the two com-pounds in the tests against the smaller numberof cells (106 cells/ml).Bacteriolytic effects. The comparative rates

of lysis of P. aeruginosa NCTC 10662 by azlo-cillin and ticarcillin are illustrated in Fig. 1. Atthe higher concentrations tested, 2,500 and 5,000jig/ml, azlocillin produced a degree of inhibitionof growth of the organism, but there was noevidence of bacteriolysis. At a lower concentra-tion, 1,000 ,ig/ml, azlocillin produced little effect,and the growth of the culture was similar to thatof the untreated control culture. In contrast,ticarcillin caused rapid lysis of P. aeruginosa

AZLOCILLIN AND TICARCILLIN 183

TABLE 1. MICs of azlocillin and ticarcillin againstP. aeruginosa NCTC 10662, P. aeruginosa ATCC

27853, and P. aeruginosa 11

P. aeruginosa Inoculum MIC (.tg/ml)Strain (cells/mi) Azlocflin Ticarcillin

NCTC 10662 3.4 X 108 1,000 62.53.4 X 106 4.0 16

ATCC 27853 3.2 X 108 >1,000 1253.2 X 106 4.0 16

11 3.7 X 108 1,000 62.53.7 X 106 4.0 31.3

100- Control A 1000

90-

80A

Z 70-a)0 60-

~~~~~50~ ~ ~ ~~ 5

40

30-

0 1 2 3 4Hours

FIG. 1. Comparative rates of lysis ofP. aeruginosaNCTC 10662 by azlocillin (A) and ticarcillin (T).Figures are concentrations in micrograms per milli-liter.

NCTC 10662 at concentrations from 250 to 1,000jig/ml. The onset of lysis was dependent uponconcentration, so that after addition of 250 jig ofticarcillin per ml, growth of the culture contin-ued at the same rate as the control culture for60 min, after which time rapid lysis occurred,whereas at a higher concentration (1,000 jig/ml),onset of lysis occurred after about 30 min.

Bactericidal effects. The bactericidal activ-ities of azlocillin and ticarcilin against logarith-mic-phase shake cultures of P. aeruginosaNCTC 10662, P. aeruginosa 11, and P. aerugi-nosa ATCC 27853 are shown in Fig. 2. Azlocillinfailed to reduce the viable counts of any of thethree strains at a concentration of 1,000 jig/mlduring the 4-h duration of the test. A higherconcentration, 2,500 jig of azlocillin per ml, wasalso relatively ineffective, and there was only asmall reduction in the viable counts of the cul-tures of P. aeruginosa NCTC 10662 and ATCC27853 at 4 h. In contrast, ticarcillin demonstratedpronounced bactericidal activity against the-three strains of P. aeruginosa, reducing the vi-

184 WHITE, COMBER, AND SUTHERLAND

P. aeruginosa NCTC 10662 able count by 90 to 99% within 4 h. The onset ofIO, bactericidal activity was concentration depen-

dent in the cases of P. aeruginosa NCTC 10662and P. aeruginosa 11. That is, at 250 ug of

109 r ticarcillin per ml, a rapid bactericidal effect wasseen from 2 h onwards; at 500 Ag/ml, the fall incount occurred between 1 and 2 h; and at 1,000

18 , u g/ml, the fall in count occurred from 30 min10 > \ tonwards. In the case of P. aeruginosa ATCC

27853, the three concentrations of ticarcillintested produced similar effects, and reduction in

07- \ \ \the viable count began from 30 min onwards.In a second bactericidal study with P. aerugi-

nosa NCTC 10662, the test was continued for 24

16 h (Fig. 3), and the antibiotic concentrations pres-ent at 3, 6, and 24 h were measured by microbi-ological assay. As in the previous tests, the bac-tericidal effects produced by azlocillin weremuch less than those demonstrated by ticarcil-

0P. aeruginosa ATCC 27853 lin. At 3 h, the viable counts in cultures contain-10- ing azlocillin (250 and 1,000 ,g/ml) were approx-

imately 107 cells/nil compared with counts of 3x 105 to 5 x 105 cells/ml in cultures containing

09p- /similar concentrations of ticarcillin. By 6 h, thecounts in the azlocillin-containing cultures hadfallen to 3 x 106 to 5 x 106 cells/ml, but resump-

o8 tion of regrowth occurred at some time after,and at 24 h the viable counts had risen to 109

E 4 \ cells/ml. With ticarcillin, on the other hand, theo 4 viable counts had fallen to about 3 x 104 cells/

1 1- ml at the end of the test, and there was noevidence of regrowth of any of the cultures.Microbiological assay showed that there wassignificant loss of activity of azlocillin in thepresence of P. aeruginosa NCTC 10662 com-pared with the inactivation which occurred insterile broth incubated at 37°C. For instance, inthe cultures containing P. aeruginosa NCTC

P. aeruginosa 11 10662, the azlocillin concentrations had fallen to10 - approximately 50% of the initial concentrations

at 3 h, only about 2% of the compound remainedat 6 h, and at 24 h there was no detectable

109- azlocillin (<0.5%). In contrast, there was no sig-nificant loss of ticarcillin activity during thecourse of the test. In a test with lower concen-trations of the penicillins (25 and 100 ,Ag/ml)

108- (Fig. 4), azlocillin failed to reduce the viablecounts, and at 24 h the growth of P. aeruginosaNCTC 10662 in both azlocillin-treated cultures

107- - \_ was the same as that of the untreated controlculture. At similar concentrations, the onset ofbactericidal activity of ticarcillin was dose re-

106 lated, so that at the higher concentration (100

FIG. 2. Comparative bactericidal activities ofazlocillin and ticarcillin against P. aeruginosa. Sym-

,_____,_____,_____,___ bols: *, control; El, aziocillin, 1,000 pg/ml; A, azlo-1 2 3 4 cillin, 2,500 pg/ml; V, ticarcillin, 250 pg/ml; *, ticar-

Hours cillin, 500 pg/ml; U, ticarcillin., 1,000 ug/mi.

ANTIMICROB. AGENTS CHEMOTHER.

VOL. 18, 1980

E(-,

c

=30

.0

HoursFIG. 3. Comparative bactericidal activities of

azlocillin and ticarcillin against P. aeruginosaNCTC 10662. Symbols: *, control; V, azlocillin, 250pg/ml; El, azlocillin, 1,000 pug/ml; V, ticarcillin, 250lug/ml; , ticarcillin, 1,000 pg/mL

E

a')coa-)

Ct

.0

HoursFIG. 4. Effects of low concentrations of azlocillin

and ticarcillin on the growth ofP. aeruginosa NCTC10662. Symbols: *, control; A, azlocillin, 25 ug/ml;El azlocillin, 100 pg/ml; A, ticarcillin, 25 plg/ml; ,

ticarcillin, 100 pg/ml.

,ug/ml) there was a distinct fall in the viablecount by 6 h, whereas there was no reduction inthe viable count in the culture containing 25 pugof ticarcillin per ml until some time later. How-ever, at 24 h the bactericidal effects of ticarcillinat both concentrations were the same, and the

AZLOCILLIN AND TICARCILLIN 185

bacterial counts fell to 2 x 105 cells/ml, a reduc-tion in the viable count of 99.9%.Morphological effects. Microscopic obser-

vations of shake cultures of P. aeruginosaNCTC 10662 revealed that azlocillin producedlong cell or filamentous forms at all concentra-tions tested up to 2,500 ,ug/ml. For instance, theeffects produced by a concentration of 1,000 Agof azlocillin per ml are illustrated in Fig. 5. After2 h, the elongated cells were 4 to 10 cells longand appeared undamaged, but at 4 h, when thefilaments were about 20 to 40 cells in length,there was evidence of lysis of a proportion of thefilamentous cells. However, at 24 h most fieldscontained only large numbers of normal shapedcells, but in a few fields a small number (<1.0%)of filnentous cells were also present, some ofwhich were in the process of division with theformation of normal cells.The effects produced by ticarcillin on the mor-

phology of P. aeruginosa NCTC 10662 werequite different from those observed with azlocil-lin. For instance, in the presence of 250 jig ofticarcillin per ml, elongated cells (averagelength, 2 to 6 cells) with bulges or spheroplastswere observed at 60 min (Fig. 6a), and the num-ber of cells (2 to 8 cells long) with spheroplastsincreased notably by 90 min (Fig. 6b). There wasevidence of extensive lysis by 3 h (Fig. 6c),although a few normal cells were also present,and at 24 h only cell debris was seen (Fig. 6d).At a higher concentration (500 jig of ticarcillinper ml), some cell elongation (2 to 4 cells long)was evident at 60 min, but a significant propor-tion of the cells showed spheroplast formation(Fig. 6e), and there was rapid lysis of the culturefrom 90 min onwards. In the culture ofP. aerugi-nosa NCTC 10662 containing 1,000 ,ug of ticar-cillin per ml, there was no significant cell elon-gation, and the fornation of spheroplasts wasobserved within 30 min (Fig. 6f). This culturelysed rapidly, and cell debris only was seen from60 min onwards.

DISCUSSION

The three cultures studied here were selectedas being typical carbenicillin-susceptible strainsof P. aeruginosa, and the results of the serialdilution tests to measure MICs, in which azlo-cillin was very active against a dilute inoculumbut relatively ineffective against a large inocu-lum, were in keeping with reports in the litera-ture describing the inoculum effect with urei-dopenicillins (3, 6, 15). The effect of inoculumsize can probably be attributed to the instabilityof azlocillin to the fl-lactamase of P. aeruginosa(2), and in the bactericidal tests reported here,in which large inocula were employed to enable

186 WHITE, COMBER, AND SUTHERLAND

FIG. 5. Effects of aziocillin (1,000 pg/ml) on the morphology of P. aeruginosa NCTC 10662 after (a) 2 h, (b)4 h, and (c) 24 h. Differential interference phase contrast (magnification, xl,)100).

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AZLOCILLIN AND TICARCILLIN 187

FIG. 6. Effects of ticarcillin on the morphology of P. aeruginosa NCTC 10662. (a) 250 pg/ml at 60 min; (b)250 Lg/ml at 90 min; (c) 250 pg/ml at 3 h; (d) 250 pg/ml at 24 h; (e) 500#)g/ml at 60 min; (f) 1,000 pg/ml at 30min. Differential interference phase contrast (magnification, x825).

lytic and morphological effects to be studied, itwas inevitable that inactivation of the ureido-penicillin should occur during the course of thetests.However, the data also show that significant

differences exist in the bactericidal effects dem-onstrated by azlocillin and ticarcillin against P.aeruginosa, apart from those that might be at-tributed to ,8-lactamase instability. For instance,in the bacteriolytic tests, the ureidopenicillinfailed to cause any significant lysis of the testorganism during the course of the test, even in

the early stages when high concentrations werepresent, whereas ticarcillin produced rapid lysis,the onset of lysis being dependent upon theconcentration of penicillin present in the culture.These differences were observed also in the vi-able count studies, in which azlocillin, at rela-tively high concentrations, brought about onlya small reduction in the bacterial numbers aftera delay of 3 h or longer. The bactericidal effectlasted for a short time only, after which regrowthoccurred and the viable counts of azlocilhin-treated cultures became the same as the con-

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188 WHITE, COMBER, AND SUTHERLAND

trols. In contrast, ticarcillin produced rapid bac-tericidal effects in keeping with the lytic effectsdescribed above, significant reductions in theviable counts were observed over a wide rangeof concentrations, and there was no evidence ofregrowth of the organisms during the tests.The activities demonstrated by the two peni-

cillins in the bacteriolytic and bactericidal testswere consistent with the different effects pro-duced on the morphology of the test strains ofP. aeruginosa. With azlocillin, long cell or fila-mentous cells were observed at most concentra-tions, and there was evidence only of transientlysis of these cells occurring after 3 or 4 h atconcentrations of 1,000 and 2,500,ug of azlocillinper ml. At lower concentrations, there was noevidence of lysis of the filaments. As incubationcontinued, the bacterial filaments were replacedby normal cells, which initially arose from divi-sion of the filaments. It seems likely that theresumption of normal growth from the filamen-tous population occurred as a result of inactiva-tion of the antibiotic by the fi-lactamase activityof the organism. At a higher concentration,10,000,ug of azlocillin per ml, there was evidenceof both cell elongation and spheroplast forma-tion, and the morphological effects producedwere reminiscent of those observed in culturescontaining 250 ,Ig of ticarcillin per ml.

In the case of ticarcillin, the extent of kill wasuniform over a wide range of concentrations inthat, in the presence of25 to 1,000 ,Ig ofticarcillinper ml, there was a fall in the viable counts ofthe cultures of about 99.9% after 24 h of incu-bation. However, the onset of bactericidal activ-ity was obviously dose related and coincidedwith the appearance of spheroplasts and subse-quent lysis. For instance, at 250 ,ug/ml there wasevidence of cell elongation in the initial incuba-tion period, and spheroplasts were seen in themajority of cells from 90 to 120 min after addi-tion of the antibiotic. At a higher concentration,500 ,ig/ml, cell elongation was less, and sphero-plasts were prominent from 60 to 90 min,whereas at a concentration of 1,000 Ig/ml, therewas no cell elongation, and spheroplast forma-tion and lysis occurred from 30 min onwards.The morphological response, filament formationwith azlocillin, and spheroplast production byticarcillin were similar in veal infusion contain-ing human serum (50% [vol/vol]).These observations suggest that the differ-

ences in the bactericidal effects seen with azlo-cillin and ticarcillin are due primarily to dose-related differences in inhibition of cell wall syn-thesis and secondarily to the /3-lactamase insta-bility of azlocillin. Further studies will be re-quired, particularly in vivo, to assess any possi-

ble clinical significance of the differences of thebactericidal activities of azlocillin and ticarcillinagainst actively growing cultures of P. aerugi-nosa.

ACKNOWLEDGMENTWe wish to thank Karen Griffin for skilled technical assist-

ance.

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