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Effects of chloramphenicol on Pseudomonas aeruginosa

by

Jean-François Léger

A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfilment

of the requirements for the degree of Master of Science.

Department of Microbiology Macdonald College of McGill University Montréal, Québec.

C Jean-François Léger, 1991

July 1991

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ABSTRACT

M.Sc. Jean-François Léger Microbiology

Effects of chloramphenicol on Pseudomonas aeruginosa

The characteristics of the effects of chloramphenicol on

Pseudomonas aeruginosa wer.e examined. Resistant strains were

easily isolated following a single passage in chloramphenicol

at 150 ~g/ml to 500 ~g/ml. Drug detoxification or altered

sensitivity of the target site could not be the mechanism of

resistance. This resistance to chloramphenicol was correlated

with the addition of an outer membrane protein with a molecu­

lar weight of 49 kDa and the loss of two outer membrane prot­

eins, one with the molecular weight of 19 kDa and the other

of about 10 kDa. The highly specifie requirement of the

resistant strains for Ca2+, Mg2 +, Mn 2+ or Sr2+ described by

Irvin and Ingram (1982) was confirmed by the observation that

the o~ter membrane of the resistant cells contained twice as

much Mg2t cation as the sensitive cells. Many other experi­

ments designed to observe the effects of chloramphenicol on

the oute~ membrane of P. aeruginosa failed. It was concluded

that the observations made in this study strongly sugg~sted a

"re-structuring" of the outer membrane of P. aeruginosa,

rendering the resistant cells more impermeable to

chloramphenicol.

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M.Sc. Jean-François Léger Microbiologie

Les effets du chloramphénicol sur Pseudomonas aeruginosa

Les caractéristiques de l'effet du chloramphénicol sur

P. aeruginosa ont été examinées. Les souches résistantes à

des concentrations de chloramphénicol variant entre 150 et

500 ~g/ml furent isolées après un seul passage sur

chloramphénicol. La détoxification de l'antibiotique ou

l'altération de la sensibilité du site d'action n'était pas

le mécanisme de résistance. La résistance au chloramphénicol

était reliée à l'addition d'une protéine de la membrane

e~terne avec une masse moléculaire de 49 kDa, et à la perte

de deux protéines de la membrane externe, une d'une masse

moléculaire de 19 kDa et l'autre d'environ 10 kDa. Le besoin

accru et spécifique des souches résistantes pour les ions

Ca2t, Mg2t

, Mn2t ou Sr2t, tel que décrit par Irvin et Ingram

(1982), a été confirmé par l'observation que la membrane

externe des souches résistantes contenait deux fois plus

d'ions de Mg2t que les souches sensibles à l'antibiotique.

Beaucoup d'autres expériences conçues pour observer les

effets du chloramphénicol sur la membrane externe ont échoué.

Il a été conclu que les observations faites lors de cette

étude suggèrent fortement une "re-structuration" de la

membraLe externe de l'organisme P. aeruginosa, rendant les

souches résistantes plus imperméables au chloramphénicol.

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1 ACINOMLZDGBMKNTS

The author expresses his appreciation to Dr. J.M. Ingram

for his interest, supervision and helpful criticism during

the course of this study.

In addition, thanks is expressed to aIl graduate

students in the department for their valuable assistançe and

encouragement and more specifically to F. Pietrantonio and J.

Kearvell.

Also, gratitude is offered to Mr. P. Levert for his

assistance in photography and the maintenance of the

e~lipment, as weIl as, to Mrs. M. Parkinson and Mrs. E. Magee

for handling all the paperwork.

The authoI is indebted to his future wife, Brigitte

Brunet, for her love, encouragement and financial support.

This book i5 dedicated to her.

The author thanks his family for their support and

encouragement.

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'l'ABU or CON'l'l:H'1'S

Page

LIST OF TABLES ..................•.................•.. viii

LIST OF FIGURES ...........•......................•... ix

INTRODUCT ION .•....••......•....••....••... '"' . . . . . . • • • • 1

LITERATURE REVIEW .......••.....••...........•..•..•.. 3

Chloramphenicol resistance .•.................... 3 Mode of action of chloramphenicol ....... ... 3 Major mechanisms of resistance against chloramphenicol ............................ 5

Chloramphenicol acetyltransferase (CAT) (drug dt!toxification) ..............•.. 5 Target site alterations ........... .... 6 Chloramphenicol uptake in Pseudomonas aeruginosa ................... " ........ .

Isolation and characterization of chloramphenicol resistant Pseudomonas aeruginosa ............... .

Isolation of chloramphenicol resistant Pseudomonas aeruginosa .................... . Physiological and biochemical character­istics of the chloramphenicol resistant

7

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cells ...................................... 12 Chloramphenicol inactivation ......•... 12 Amino acids transport ..............•.. 13

Divalent cation regulation of chloramphen-icol resistance in Pseudomonas aeruginosa .. 13

Major outer membrane proteins of Pseudomonas aerug~nosa ...................................... 16

Forin proteins ............................. 16 Lipoproteins .................. ço • • • • • • • • • • •• 20 Differences ln antibiotic resistant Pseudomonas aerug~nosa .................•.•. 21

Spheroplast formation in Pseudomonas aeruginosa. 23 Alkaline phosphatase enzyme •..........•......••. 24

MATERIALS AND METHODS •••••••••••••••••••••••••••••••• 26

Microorganisms ..............•.........•......... 26 Chemicals ....................................... 26 Medi a ........................................•.. 27 Growth conditions ..........•.................•.. 27 Isolation of chloramphenicol resistant strains .• 28 Effects of chloramphenicol on outer-membrane .•.• 28

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Spheroplast formation experiment ......••... 28 Penicillin G experiment .................... 29 The release of alkaline phosphatase ........ 29 Binding of chloramphenicol to

P. aeruginosa ............................. 30 Separation and characterisation of the outer membrane ................................. 31

Separation of the outer membrane (sucrose gradient) ........................ 31

Estimation of prote in concentration ........ 32 12% Slab sodium dodecyl sulfate polyacrylamide gel electrophoresis ........ 32

Vertical slab gel ................•.... 33 Electrode buffer ...................... 33 Digestion buffer ...................... 33 Running conditions .................... 34 Protein standards ................•.... 34 Fixing and staining the proteins

in the gels .......................... 34 Atomic absorption (Mg+ 2 concentration) ...... 35

Respiration activity of whole cell suspension ... 36 Preparation of whole cell suspension ....... 36 Substrates and equipment ................... 36 Respiration assays ......................... 37

RESULTS ........................•..................... 38

Effects of chloramphenicol on the outer membr ane ..................•..................... 38

Spheroplast formation experimen~ ........... 38 penicillin G experiment .................... 39 The release of alkaline phosphatase ........ 46 The binding of chloramphenicol to Pseudomonas aeruginosa ..................... 48

Separation and characterization of the outer membr ane ........................................ 4 9

Outer membrane protein profiles ............ 49 Mg2+ concentration in the outer membrane .... 52

Respiration activity of whole cell suspension ... 54

DISCUSSION... . .... . ...... . . .... . .... . . . . .. . ...... . . .. 56

BIBLIOGRA.PHY ..•......•.........•..................... 63

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LIST Oi' TABLBS

Table

1 Sorne major outer membrane protejns of P. aeruginosa ................................. 17

2 Respiration in 9027 and RC150/9027 .....••.••.• 55

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Figures

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LIST 01' FIGURES

Structure of chloramphenicol

Growth of PU21 RC150 in nutrient broth

Growth of PU21 RC500 in nutrient broth

Effect of Mg2+ on growth and resistance in

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citrate Min P ................................................... 15

Growth of PU21 in nutrient broth (exposed to 150 ~g/ml chloramphenicol and 25 ~g/m1 penicillin G) ................................. 41

Growth of RC150 in nutrient broth (exposed to 150 ~g/ml chloramphenicol and 25 ~g/ml penicillin G) ................................. 42

Growth of PU21 in nutrient broth (exposed to 500 ~g/ml chloramphenicol and 100 ~g/ml penicillin G) ••••••••••••••••••••••••••••••••• 44

Growth of RC500 in nutrient broth (exposed to 500 ~g/ml chloramphenicol and 100 ~g/ml penicillin G) ••••••••••••••••••••••••••••••••• 45

Alkaline phosphatase (APase) activity in 9027 ................................................................................ 47

Twelve percent SDS-PAGE of outer membrane from chlorarnphenicol resistant and sensitive strains of P. aeruginosa .............................. 5 a Mg2+ concentration in the outer membrane of PU21, RC150 and RC500 ......................... 53

1

INTRODUCTION

Pseudomonas aeruginosa is a gram-negative bacterium that

can be found almost anywhere, more especiaIIy in soil. The

organism produces a blue phenazine pigment, pyocyanine

(Ingram and BIackwoad, 1962). This pigment i5 generally used

to classify P. aeruginosa. Chloramphenicol resistant P.

aeruginosa do not produce this characteristic phenazine

pigment, pyocyanine (Ingram and Hassan, 1975). New

classification procedures such as serological responses have

been developed in order to circumvent this problem.

Bacteraemia and a high mortali ty rate due to P.

aeruginosa infection were frequent during the introduction of

antibiotic chemotherapy (Finland et al., 1959; Reynolds et

al., 1975). The prolonged and sometimes unnecessary

prescription of antibiotics gradually helped colonization by

P. aerug.inosa. This bacteria is extremely resistant to many

antibiotics. Even with the development of more potel.t

antibiotics, P. aeruginosa continues ta maintain its

intrinsic resistance, especially to chioramphenicoi (Gottlieb

et al., 1948; Landerkin et al., 1950; Hennberg and Muller,

1951) .

The outer membrane of gram-negative bacteria, such as P.

aeruginosa, provides a permeability barrier which makes the

bacteria less permeable than gram-positive organisms ta a

variety of molecules including disinfectants, drugs and

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enzymes (Lieve, 1974). The outer membrane of P. aeruginosa

provides compartmentalization of catabolic enzymes and is a

selective barrier for other vital solutes. There is a

different mechanism for solutes to cross the outer membrane:

specifie and non-specifie pores, specific receptor complexes

and during special conditions by a hydrophobic pathway

(Nikaido and Vaara, 1985). This study was undertaken so that

we could learn more about the effects that chloramphenicol

has on P. aeruginosa and the resistanee meehanism the

organism develops against this antibiotic.

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LITERATURE RBVIEW

CHLORAMPHENICOL RESISTANCE

Mode of action of chloramphenicol

Chloramphenicol is a broad-spectrum antibiotic produced

by Streptomyces venezuelae and, more practically, by chemical

synthesis. It has a simple chemical structure (Rebstock et

al., 1949) as shown in Fig. 1. Of four possible

stereoisomers, only one possesses antibacterial activity, D-

threo-chloramphenicol. Chloramphenicol inhibits prote in syn­

thesis by weakly binding to the 50S ribosomal subunit

(Pestka, 1975). The peptidyl transferase reaction centre

seems to be the site of action of this antibiotic. The exact

mechanism of this peptide bond formation inhibition remains

unclear (Pestka, 1977). Although eukaryotic cells are gen-

erally resistant to the effects of the antibiotic, it is

usually associated with toxic side effects. These toxic side

effects are at least partly due to the fact that

mitochondrial ribosomes (and chloroplasts) are sensitive to

chloramphenicol (Pe~tka, 1975). Under normal conditions,

growth and protein synthesis of bacterial calls are inhibited

by the same concentration of chloramphenicol either in vivo

or in vitro, suggesting that chloramphenicol is easily taken

in by the sensitive cells .

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Figure 1

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Fig. 1. Structure of chloramphenicol.

Adapted from Irvin (1983).

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"

o H " \,/C-CHCI2

~ ~ ~ C-C-C-OH 1 1 1 OH H H

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Major mechanisms of resistance against chloramphenicol

The majority of resistance phenomena demonstrated to

date appear to involve 3 major types of mechanism: drug

detoxification, target site alteration and interference with

drug transport. These mechanisms are discussed in more detail

in Sykes and Matthew (1976), Chopra and Howe (1978) and

Davies and Smith (1978). A brief review of these mechanisms

will be discussed below.

Chloramphenicol-acetyltransferase (CAT)

(drug detoxi fication)

Many natural isolates (mainly enterobacteria) were found

to possess enzymes which catalysed the modification of

antibiotics and antibacterial agents 50 that they are no

longer capable of affecting the growth of bacterial cells.

These enzymes have been identified and characterized. They

were found to be almost exclusively encoded by extra­

chromosomal genes with the exception of sorne B-lactamases

(Davies and Smith, 1978).

In the presence of acetyl-coenzyme A the R-factor

strains of Escherichia coli were reported to inactivate

chloramphenicol (OkamoLo and Suzuki, 1965). The CAT enzyme

inactivated chloramphenicol by 3-Q-acetylation to yield 3

acetyl and 1,3-diacetyl-chloramphenlcol (Suzuki and Okamoto,

1967; Mise and Suzuki, 1968). These enzymes have since been

detected in a wide variety of bacterial genera (Fitton et

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al., 1978). It was aIse found that the use of the enzyme CAT

by Pseudomonas as a mechanism of resistance was less frequent

than other Gram negative genera (Mills and Holloway, 1976).

AIso, even if the CAT enzyme was found in Pseudomonas, the

activity of the enzyme could not account for the exceptional­

ly high level of resistance (Mills and Holloway, 1976). Irvin

(1983) also showed that chloramphenicol acetylation was very

low in the P. aeruginosa strains used in this study and that

the CAT activity could not account for the high level of

resista~ce observed during these experiments.

Target site alterations

It appears that no evidence has been documented whereby

chloramphenicol resistance includes target site alteration in

P. aeruginosa. Only one report involves ribosomal protein

alteration. Chloramphenicol resistant mutants of Bacillus

subtilis showed reduced binding of chloramphenicel to the

ribosomes (Osawa et al., 1973). The mutation was induced by

nitrosoguanidine treatment and the level of resistance of

this mutant was very low (5 ~g/ml). No assays were made on

the biological activity of chloramphenicol on cell-free

protein synthesis. Another report (Baugham and Fahnestock,

1979) utilizing site-directed mutagenesis demonstrated the

isolation of a chloramphenicol resistant mutant of E. coli.

Although analysis indicated that the mutation was mapped in

the ribosomal gene cluster these authors were not able to

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demonstrate any alteration in the target site bindlng of

chloramphenicol. The nature of this resistance remains

unclear since chloramphenicol inactivation and reduced per­

meability were ruled out.

Chloramphenicol uptake in Pseudomonas aeruginosa

7

So far, no researchers were able to directly dernonstrate

the accumulation of chloramphenicol into P. aeruginosa.

Vasquez, 1963; Gaffney et al., 1981; Irvin, 1983 were not

able to measure the uptake of 14C-chloramph9nicol either by

standard filtration transport assays or by rapid

centrifugation techniques, even in sensitive cells. In fact,

14C-chloramphenicol was found to bind to filters and the

radioactivity on these filters was meaningless and bore

absolutely no relationship to the concentration of

chloramphenicol in the medium, cell concentration or

incubation time. More recently, two researchers (Abdel-Sayed,

1987; Burns et al., 1989) claimed to have indirectly measured

the accumulation of chloramphenicol in sensitive cells of

Pseudomonas. Instead of directly measuring the uptake of

chloramphenicol, they measured the depletion of the

antibiotic in the medium. Abdel-Sayed suggested a biphasic

kinetic accumulation of chloramphenicol into P. aeruginosa, a

rapid uptake which lasted for 1 min. followed by a slower

uptake which continued for 5 minutes. Burns demonstrated the

accumulation of 14C-chloramphenicol into sensitive

8

Pseudornonas cepacia by again indirectly measuring the

depletion of chloramphenicol from the medium. However, he

clearly showed a pronounced decrease in uptake of

chloramphenicol into resistant strains of P. cepacia. He

concluded that this decreased permeability was a mechanism of

resistance in P. cepacia.

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ISOLATION AND CHARACTERIZATION OF CHLORAMPHENICOL RESISTANT

PSEUDOMONAS AERUGINOSA

This section is a mini-review of Irvin (Ph.D. Thesis,

McGill University, 1983). For a more detailed discussion

beyond the scope of this review, please refer to the original

the~,is .

Isolation of chloramphenicol resistant Pseudomonas aeruginosa

The isolation of resistant bacterial strains usually

requires several growth cycles in increasing concentrations

of the antibacterial agent. This "training" is usually needed

aga in st common therapeutic agents (Pitton, 1972). Ingram and

Hassan (1975) demonstrated that P. aeruginosa could become

resistant to high levels of chloramphenicol in only one step.

The exposure of wild-type strains to high levels of

chloramphenicol resulted in the outgrowth of a resistant

population. A brief period of growth inhibition was observed

i . e. 10-12 h for 150 J.Lg/ml chloramphenicol (RC150) and about

24 h for 500 J.Lg/ml chloramphenicol (RC500).

Figure 2 illustrates the growth of PU21/RC150 in nutri-

ent broth (Irvin, 1983). The addition of 150 J.Lg/ml of

chloramphenicol did not significantly affect the growth of

RC150. The increased resistance of RC500 is reflected in

considerably less growth inhibition in chloramphenicol at 500

~g/ml (Fig. 3). Compared to the parent strains, the growth

rates and final yields are reduced (Irvin, 1983). This high

10

Figure 2

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Fig. 2. Growth of PU21 RC150 in nutrient broth.

Symbols: (.) PU21

(.) RC150 minus chloramphenicol (0) RC150 plus chloramphenicol (150 ~g/ml) (0) RC150 plus chloramphenicol (500 ~g/ml)

Adapted from Irvin (1983).

l

~ ~ ·10 o

·01· --t-2 --{-~6~-t---d---­TIME [h]

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Figure 3

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Fig. 3. Growth of PU21 RC500 in nutrient broth.

Symbols: (0) PU21 ( .) RC500 plus chloramphenicol (150 J.L9/ml) (_) RC500 plus chloramphenicol (500 J.L9/ml)

Adapted from Irvin (1983).

...... .......

-

/

o--a-o -0 -0 o /..--____ -0 1 /" ...---...-1=. oA

~ •

'~---i2~-i4---i6---t8---+--10

TlME [h]

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level of resistance of RC150 and RC500 is very stable. These

strains retain the high resistance characteristics even after

5 to 9 growth cycles (from lag to stationary) into drug-free

broth (Irvin, 1983).

Physiological and biochemical characteristics of the

chloramphenicol resistant cells.

Chloramphenicol inactivation

The enzyme chloramphenicol-acetyltransferase (CAT) was

assayed by Irvin (1983) in both wild type PU21 and

chloramphenicol resistant strains (RC150 and RC500). The

assays were conducted both in vitro, using cell-free extracts

and in vivo, using whole cells. A very low concentration of

chloramphenicol-l-acetate was detected after 2 h of incuba­

tion with highly concentrated cell-free extracts. This low

level of CAT activity was detected in aIl strains and at aIl

times. The age of the cultures and the presence, or absence,

of the drug did not influence this low level of acetylation.

These observations suggested that the CAT activity is not

regulated by the drug or the physiology of the cells. This

level of acetylation is insufficient to explain the high

level of resistance observed in P. aeruginosa (Irvin, 1983).

The amount of free chloramphenicol in the media was

assayed by a spectrophotometric assay (Irvin, 1983). The

results indicated that only a trivial amount of

chlorampheniccl was net available for the reaction. This

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reduction was insignificant and indicated that essentially

aIl the drug remained free in the medium (Irvin, 1983).

Amino acids transport

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The rate of amine acid uptake was measured in wild type

and chloramphenicol resistant variants of P. aeruginosa

(Irvin and Ingram, 1980). The initial rate and steady-rate

levels of amine acid uptake were substantially reduced in the

resistant variants. However, a dramatic effect was observed

in cells grown in the presence of chloramphenicol. The

accumulation of amine acids was highly depressed. This

reduction of accumulation is not a function of depressed

in:orporation (Irvin and Ingram, 1980). These results suggest

that chloramphenicol resistance in P. aeruginosa is

associated with alterations in the permeability of the

membranes (Irvin and Ingram, 1980). They also indicate that

the physiology of the resistant cells can be influenced by

the presence of the drug.

Divalent cation regulation of chloramphenicol resistance in

Pseudomonas aeruginosa

Irvin and Ingram (1982) reported that the growth

characteristics and the Ievel of resistance of the

chloramphenicol resistant strains of P. aeruginosa, varied

widely as a function of the composition of the growth media.

SpecificaIly, when the resistant strains were grown on

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substrates, which function as efficient chelators of divalent

cations, a reduction of growth rate and an increased

sensitivity to chloramphenicol was observed. They suggested

that this requirement for diva lent cations may be

prerequisite for the expression of chloramphenicol

resistance. They analyzed the growth patterns and resistance

levels under different concentrations of divalent cations

(Fig. 4). It was demonstrated that chloramphenicol resistance

is dependent on the concentration of divalent cations (Irvin

and Ingram, 1982). Different diva lent cations were tested but

Mg2+, Mn2+, Ca2+ or Sr2t were found to be essential.

These results again suggest strongly that the envelope

exerts a critical influence on the expression of

chloramphenicol resistance in P. aeruginosa probably by

making the outer cell wall more impermeable to toxic agents

such as chloramphenicol.

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Figure 4

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Fig. 4. Effect of Mg2+ on growth and resistance in citrate

Min P.

Cultures of (A) PU21 RC150 (±chloramphenicol at 150 ~q/ml) and (B) PU21 RCSOO (±chloramphenicol at 500

~q/ml) were qrown in citrate Min P supplemented with various concentrations of MgS04 as follows: (~ , 0 ) 20 mM Mg2+

(a , c ) 1 mM Mg2+

( •• â ) 100 J1M Mg2+

Closed symbols - minus chloramphenicol. Open symbols - plus chloramphenicol.

1-' ..... . ~

.. ..

OPTICAL DENSITY (660 nm)

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MAJOR OUTER MEMBRANE PROTEINS OF PSEUDOMONAS AERUGINOSA

(Hancock et al., 1990)

The outer membrane of P. aeruginosa is involved in the

exclusion of potentially harmful substances such as

detergents, disinfectants, ... But often molecules, su ch as

substrates, must cross this barrier. They can enter cells by

several different paths, such as, specifie and non-specifie

pores, specifie receptor complexes and by a hydrophobie

pathway (Nikaido and Vaara, 1985). In order for produets to

be eKc~eted from cells, they must also cross the outer

membrane, so that ~t must also be involved in secretion of

molecules (Hirst and Welch, 1988). AlI of these functions are

assumed to be conducted or assisted by the outer membrane

proteins (Opr). Sorne of these proteins will be briefly

reviewed below (Table 1) .

Porin proteins

In P. aeruginosa, porin preteins are noneovalently, but

strongly associated with peptideglycan (Hancoek et al.,1981).

They are heat modifiable and are resistant to sodium dodecyl

sulfate d~naturation. They form water-filled pores which are

located throughout the width of the bilayer outer membrane

and allow non-specifie diffusion of solutes. The monomer

moleeular weights range from 28 000 to 48 000 and are active

as oligomers (Hancock, 1987).

Perin protein P (Opr P) is induced under low phosphate

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Table 1. Sorne major outer membrane proteins of P. aeruginosa

Apparent molecular protein weight on SOS-page Characteristics Refs. a

P (OprP) 48 000 Induced in low 1, 2, phosphate 3, 4

Involve in phos-phate transport

Dl (OprB) 46 000 Induced in glucose Glucose porin

E (OprE) 44 000 porin

F (OprF) 38 000 Constitutive porin/ structural

H2 (OprL) 20 500 Constitutive Structural/lipo-

prote in

l (OprI) 9 000 Constitutive 5, 6, Structural/lipo- 7

protein

a. Reference numbers refer to the following papers: 1, Hancock et al. (1982); 2,Worobec et al. (1988); 3,Poole and Hancock(1986b); 4,Siehnel et al.(1988); 5,Mizuno and Kageyama(1979); 6,Cornelis et al. (1989); 7,Duchêne et al. (1989) •

Adapted from Hancock et al. (1990)

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concentrations and is not present when the medium is supplied

with more than 0.15 mM of phosphate (Hancock et al., 1982).

Opr P is very important for the phosphate-specific transport

(Pst) system of P. aeruginosa (Poole and Hancock, 1986a). Opr

P was c10ned in E. coli and was shown to be regulated by the

phosphate level in the media. The E. coli phosphate-

starvation-inducible outer membrane prote in PhoE was observed

to have similar biochemical properties with Opr P (Worobec et

al., 1988). But the amine acid sequences have no similarities

(Siehnel et al., 1990b) and their functions also differ

(Hancock et al., 1986). PhoE is an anion-selective general

pore, whereas Opr P has a 10Q-fold preference for phosphate

over other anions (Hancock and Benz, 1986) which is due to a

phosphate-bindlng site which is composed of charged lysine e-

amino groups. Pre/teins homologous to Opr P were shown to

cross-react immunologically in four different Pseudomonas

species (Poole and Hancock, 1986b).

Another porin prote in is protein Dl (Opr B). Porin Dl, a

trimer, is induced by the presence of glucose in the media,

which re~ults in the induction of a high affinity glucose

uptake system and the glucose-binding protein (Hancock and

Carey, 1980). A preference for glucose and xylose over other

sugars was suggested by liposome-swelling studies (Trias et

al., 1988). Porin Dl is also heat modifiable.

A third porin prote in located in the outer membrane of

P. aeruginosa is porin protein E (Opr E). Little is known of

the outer membrane porin protein E. By liposome-swelling

techniques, Opr E was demonstrated to be a general porin

(Yoshihara and Nakae, 1989). This porin should allow the

19

passage of trisaccharides (Siehnel et al., 1990a), although a

smaller exclusion lirnit was suggested and more studies must

be done to confirm this assumption.

porin F (Opr F) is one of the most studied outer

membrane proteins and its function is currently a source of

controversy (Siehnel et al., 1990a). Sorne researchers

(Hancock et al., 1979; Benz and Hancock, 1981; Trias et al.,

1988) have suggested that a small portion of the Opr F

molecules, 400 out of 200 000 per cell, forrn large channels

that will allow the passage of saccharides up to 3 000 mol­

ecular weight. This channel is estimated to average 2 nm in

diameter (Nikaido and Hancock, 1986). The Opr F molecules

that do not forrn these channels will form srnall porins which

are predicted to be antibiotic impermeant (Woodruff et al.,

1986). On the other end, other researchers have suggested

that Opr F has a low exclusion limit and that it has no

function as a porin so that even disaccharides are excluded

(Yoneyama et al., 1986; Gotoh et al.,1989i Yoshihara and

Nakae, 1989). The methodologies used in these studies have

already been criticized (Nikaido, 1989). No reports could be

found on the discrepancies of these findings. Also, mutants

lacking Opr F have drarnatic structural alterations (Gotoh et

al., 1989). These mutants are rounded cells, slower growing

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and leak periplasmic proteins into the media. These data

suggest that Opr F plays an important role in maintaining the

integrity of the outer membrane.

Lipoproteins

In 1979, Mizuno and Kageyama (1979) discovered a

peptidoglycan-associated lipoprotein in the cell wall of P.

aeruginosa. The same band was later shown to be, with the

help of better separating techniques, two distinct outer

membrane proteins (Hancock et al., 1981). Only one of the two

proteins, outer membrane lipoprotein H2 (Opr L), was found to

be associated with the peptidoglycan even after treatments

with 2% sodium dodecyl sulfate at 35 to 75°C (Hancock et

al. , 1981). The other outer membrane protein, Hl (Opr H), is

not a lipoprotein and is not associated with the

peptidoglycan. A high magnesium concentration in the medium

represses the formation of this protein. These studies

prompted a debate concerning its absence or presence in the

outer membrane of polymyxin B resistant P. aeruginosa (Nicas

and Hancock, 1980; Gilleland and Beckham, 1982). These data

will be discussed later on in this section.

Lipoprotein l (Opr I) is a low molecular weight prote in

and is highly abundant (Mizuno and Kageyama, 1979). Opr 1 is

analogous to Braun' s lipoprotein found in E. coli (Braun,

1975), Salmonella and Serratia (Brown et al., 1970; Halegoua

et al., 1974). Sorne strains of P. aerug_inosa contain both

covalently and non-covalently peptidoglycan-associated OprI

(Mizuno and Kageyama, 1979). PA01 contains only non­

covalently associated Opr l (Hancock et al.,1981). These

lipoproteins may help to anchor the outer membrane layer to

the peptidoglycan layer (Braun et al., 1970).

Differences in antibiotic resistant Pseudomonas aeruginosa

21

In polymyxin B resistant P. aeruginosa, Nicas and

Hancock (1980) found the outer membrane protein Hl (0pr H) to

be present in the cell wall of the resistant cells. Gilleland

and Beckham (1982) found no trace of that protein in their

resistant strains. They both agree that a high magnesium

concentration repressed the formation of Opr H and that at

the same time the organism was susceptible to polyrnyxin B

action. Nicas and Hancock (1980, 1983) thought that Opr H

replaced the magnesiurn cations in the outer membrane when the

concentration of the divalent cation was too low in the

media, thereby resulting in antibiotic and chelator resistant

P. aeruginosa. More recently, Said et al. (1987) induced the

formation of Hl without lowering the magnesium concentration

and found that the cells remdined fully sensitive to

pOlymyxin Band to th~ metal chelator, EDTA. The resistance

mechanism rnay involve the absence of magnesium cations and

sorne phospholipid modifications.

Hirai et al. (1987) observed a new outer membrane

prote in with a molecular weight of 54 kDa, in a norfloxacin

22

resistant mutant of P. aeruginosa. This mutant strain, nfxB,

had no other apparent changes suggesting that this new pro-

tein may be involved in the resistance mechanism against

norfloxacin (Harai et al., 1987). The mechanism of resistance

still remains a mystery.

50 far, only the appearance of outer membrane proteins

in resistant cells was discussed, but there is evidence of

outer membrane proteins missing in the outer membrane of

resistant cells of P. aeruginosa. This is the case of a

quir.olone resistant strain isolated during experimental

endocarditis. Chamberland et al. (1989) showed that the

altered permeability was correlated with the reduction of

outer membrane protein G (Opr G) and a loss of a 40 kDa outer

membrane protein. The resistant cells against norfloxacin and

quinolone were also "cross-resistant" to chloramphenicol

(Hirai et al., 1987; Chamberland et al., 1989).

In chloramphenicol resistant P. cepacia, a new 18 kDa

outer membrane protein was observed in the strain 249-2

(pLIN1) and was not identified in other isogenic strains

(Burns et al., 1989). This band was only visible in sorne gels

whereas other gels did not show that band. These data show

sorne of the differences in the outer membrane protein profile

due to antibiotic resistance in P. aeruginosa. The differ-

ences observed are not consIstent, but they can be inter-

pr~ted as a primary or secondary effect of a "re-structured"

outer membrane.

23

SPHEROPLAST FORMATION

Spheroplast formation can easily be induced in E. coli

by treatment with ethylenediaminetetraacetic acid (EDTA) and

by lysozyme (Malamy and Horecker, 1964 ; Birdsell and Cota­

Robles, 1967). Exposure to EDTA increases the accessibility

of the cell wall mucopeptide to lysozyme, however, it was

shown that when the same treatment was applied to P.

aeruginosa, the cells were lysed (Eagon and Carson, 1965 ;

Asbell and Eagon, 1966 ; Gray and Wilkinson, 1965). They

also demonstrated that when P. aeruginosa was treated with

non-lytic concentrations of EDTA and then treated with

lysozyme, the cells formed osmotically fragile rods termed

osmoplasts (Eagon and Carson, 1965; Asbell and Eagon, 1966).

The only true spheroplasts were observed after the cells were

exposed to 0.2 M MgCl z and then treated with lysozyme (Cheng

et al., 197 Ob). This Mg+2 exposure appears to increase the

cell wall permeability amd thus allows lysozyme to reach the

peptidoglycan layer to produce spheroplasts. Other substances

were tested for their ability to increase permeability of the

cell wall; complement (Feingold et al., 1968), polymyxin

(Brown and Melling, 1969), freezing and thawing (Wolin,

1966), growth at elevated temperature (Hoffman et al., 1966)

and mutation (Sekiguchi and Tida, 1967). In this study,

chloramphenicol is tested for its ability to increase cell

wall permeability in susceptible strains by trying to induce

spheroplast formation.

-

24

ALKALINE PHOSPHATASE ENZYME

Alkaline phosphatase has been studied in many bacteria,

such as E. coli (Torriani, 1960), Staphylococcus aureus (Shah

and Blobel, 1967), several Bacillus species (Dobozyand

Hanner, 1969), Pseudomonas fluorescens (Friedberg and Avigad,

1967) and more recently P. aeruginosa (Cheng et al., 1970b).

In P. aeruginosa, the enzyme binds to the internaI portion of

the tripartite layer of the cell wall rather then to the

cytoplasmic membrane or the peptidoglycan layer (Cheng et

al., 1971). Alkaline phosphatase is a phosphohydrolase and a

phosphotransferase, dephosphorylating and phosphorylating

monosubstituted phosphate molecules (Wyckoff, 1987). This

enzyme is induced when the cells are deprived of inorganic

phosphate (Cheng et al., 1970b) and is repressed upon the

addition of inorganic phosphate to the media. It is not known

if alkaline phosphatase has an intra-cellular function.

The physical characteristics of alkaline phosphatase

were studied in more detail in E. coli. With a molecular

weight of 94 000, the enzyme is a dimer of identical sub­

units, with 2 active sites (Bradshawet al., 1981). Each site

comprises 2 zinc ions, although 3 can be present from time to

time. Sometirnes the zinc atorns are replaced by rnagnesium or

cadmium in various ionic forms (Coleman et al., 1983). The

general structure of each monomer is an alpha-beta-alpha core

with 10 oeta ribbons passing through the monomer core. Also,

the amine acids that are at close proximity to the Metal ions

25

are histidine, aspartic acid, threonine and glutamic acid

(Coleman, 1987). Alkaline phosphatase is a globular protein

with symmetry about the subunit junction, as observed with X­

ray diffraction (Wyckoff et al., 1983).

Less work has been conducted on the physical

characteristics of alkaline phosphatase in P. aeruginosa. Day

and Ingram (1973) noted that the metalloenzyme had a molecu­

lar weight of 68 000 in a dimeric form, but a tetrameric form

also exists. Four mol of zinc were found per mol of enzyme

and the Km for p-nitro-phenyl phosphate was similar to the E.

coli enzyme (6.6 x 10-sM). A high concentration of

hydrophobie amine acids was found in alkaline phosphatase of

P. aeruginosa. In E. coli the enzyme is released from the

periplasmic space by subjecting the cells to osmotic shock

with ethylenediaminetetraacetic 3cid (EDTA) (Neu and Chou,

1967), however, EDTA lyses P. aeruginosa. When cells are

washed with 20% sucrose the enzyme that is affiliated with

the outer surface of the cell wall is released, but only O.2M

MgCl 2 will, in addition, release periplasm loeated enzy~e

(Cheng et al., 1970b). Although initially located in the

periplasmic space, alkaline phosphatase eventually migrates

through the outer membrane to be found in the culture fil­

trate. The magnesium treatment of eells gave rise to the

hypothesis that magnesium and calcium (to a lesser extent)

had an important role in the outer membrane structural integ­

rit y of P. aeruginosa.

{

26

MaTBRIALS AND MBTBODS

MICROORGANISMS

P. aeruginosa PU21, and the substrains which were resis-

tant to 150 and 500 ~g chloramphenicol per ml, PU21/RC150 and

PU21/RC500, were used in this study. The strain ATCC 9027 was

also used but to a lesser extent. Stock cultures were main-

tained on Pseudomonas P (Oifco) agar slants at room tempera­

ture. These slants were transferred weekly. AlI P. aeruginosa

strains were also stored as frozen suspensions in Tryptone-

Yeast Extract Bcoth (TYE) at -40 C. The chloramphenicol

resistant isolates were always maintained in the presence of

the drug.

CHEMICALS

The labelled 14C-chloramphenicol was purchased from New

England Nuclear Corp., Boston, Massachusetts.

Low Molecular Weight (LMW) protein standards, sodium

dodecyl sulfate (SOS), TEMED (N,N,N,N'-tetramethylethylenedi-

amine), bis (N,N'-methylenebisacrylamide), acrylamide, Bromo-

phenol blue, mercaptoethanol, Coomassie blue, and ultrapure

sucrose were obtained from the BIO-RAD Laboratories,

Richmond, California.

Unless specifically listed, the remaining reagents were

the best grade available from local commercial sources.

,-..

27

MEDIA

Different types of medium were used, depending on the

nature of the experiment. Nutrient broth (NB-Difco), prepared

according to the manufacturer's instructions, was used in the

isolation of the RC strains, in studies on the acquisition of

resistance and in routine growth and physiology experiments

(Irvin and Ingram, 1980).

For the alkaline phosphatase synthesis experiment and

the respiration assay, the glucose-ammonium salts Proteose-

Peptone medium of Cheng et al. (1970a) was used. Proteose-

Peptone (0.5%) was added as Proteose-Peptone #3 (Difco).

Irrespective of the medium employed for the cultivation

of the resistant substrains, aIl media contained the

appropriate concentration of chloramphenicol (150, 500

~g/ml). The chloramphenicol was sterilized by Millipore

filtration (0.45 ~m pore size) and was added just prior to

inoculation. AlI media were prepared using glass distilled

water.

GROWTH CONDITIONS

The organisms were grown with constant agitation, 250

rpm, in a Psychotherm Incubator Shaker (New Brunswick Scien­

tific Co.), at 37 C. Isolated colonies wele inoculated in 50

ml of medium contained in a 250 ml Erlenrneyer flask, the

inocula was then incubated for 12 h (unless otherwise indica-

ted). One percent of the inocula was then transferred into

28

fresh media. The liquid to total volume ratio was maintained

at 1:5. The growth of the cultures was followed by measuring

changes in turbidity of the culture. AIl measurements were

read at  = 660nm. The spectrophotometers were previously

blanked with sterile medium.

ISOLATION OF CHLORAMPHENICOL RESISTANT STRAINS

(Irvin and Ingram, 1980)

The parent strains were inoculated into nutrient broth

containing chloramphenicol at 150 or 500 ~g/ml. Growth was

initially inhibited but outgrowth of a resistant population

occurred within 8-24 h. The culture was then plated on

Pseudomonas P agar (Difco) containing the appropriate concen-

trations of chlorampher.icol.

EFFECTS OF CHLORAMPHENICOL ON OUTER-MEMBRANE

Spheroplast formation experiment

The cells (PU21 & 9027) were grown in the glucose-amrnon-

ium salts- proteose peptone medium as previously described

above. For the controls, 20 ml of a 14-h culture was centri-

fuged at 13 000 X 9 and resuspended into 20 ml of 0.2M

MgCI 2 '7H20 (pH 8.4) containing 0.5 mg/ml lysozy~e. The cell

suspension was incubated for 30 min at 25 C in a water bath

shaker, centrifuged, and resuspended into O.01M MgC1 2 in

O.01M Tris buffer, pH 8.4 (Cheng et al., 1970b). Another 20

ml was prepared in the same manner except that 0.2M

... ..

29

MgC1 2 ' 7H20 (pH 8.4) was replaced by 500 Ilg/rnl

chloramphenicol. This time the incubation periods were 0, 10,

20 and 30 min at 25 C. The cells were observed under a phase-

contrast microscope.

penicillin G experiment

The cells (PU21) were cultivated in glucose-ammonium

salts-proteose peptone medium for 14-h. Cells were then

centrifuged at 13 000 X 9 and resuspended into the same

medium with (or without for control) 500 Ilg/m1 of

chloramphenicol and were then incubated for 30 min at 37 C. A

5% inoculum was transferred into another flask containing (or

not) 100 Ilg/m1 of penicillin G. Growth was then monitored

spectrophotometrically at À = 660nm.

The release of alkaline phosphatase

The cells (9027) were grown for 14-h in glucose-amnlonium

salts-proteose peptone medium. Twenty ml of celis was centri-

fuged at 13 000 X 9 and either resuspended in 20 ml of 0.2M

MgCI 2'7H20 (Cheng et al., 1970a) or in 20 ml of different

concentrations of chloramphenicol in order to release the

aikaline phosphatase .

Alkaline phosphatase was assayed by the method of Neu

and Heppel (1965). The reaction mixture contained 0.7 ml of

1.0M Tris buffer (pH 8.4), 0.1 ml of 0 .lM MgCI 2 ' 7H20, 0.1 ml

of O.OlM p-nitrophenyl phosphate and 0.1 ml of enzyme extract

(

{

30

in a 1.0 ml cuvette. Activity was measured by the increase in

absorbance at 420nm. One unit of enzyme is equivalent to an

increase of 1.0 OO/minute.

Binding of chloramphenicol to Pseudomonas aeruqinosa

Both wild type 9027 and resistant substrain RC150 were

tested for their ability ta bind 14C-chloramphenicol. Cells

fram a 100 ml culture were harvested after the cells reached

an 00 reading of 1.0 by centrifugation at 13 000 X 9 for 15

min at 4 C. The cells were grown in nutrient broth medium.

These cells were then ~esuspended in nutrient broth with the

appropriate volume to give a final concentration of 0, 0.2,

0.4, 0.6, 0.8 and 1.0 00.

One ml of cells (at different concentrations) was added

to 1.0 ml of 14C-chloramphenicol in dH20. The final concent:a­

tian of 14(.-chloramphenicol in the reaction test tubes was

8.3 ~g/ml with an activity of 1.25 x 10-1 ~Ci/ml. The reac­

tian mixture (2.0 ml) was gently shaken for 15 min and

immediately filtered through 0.45 ~ Millipore filters. These

filters were prewashed with nutrient broth. A competitive

binding assay was accomplished by slowly adding 4.0 ml of

cold chlorampherdcol (40 Jlg/ml) through the filters right

after the reaction mixture. The reaction test tubes were

rinsed with 2.0 ml nutrient broth. The washed fluid was then

filtered. The filters were subsequently rinsed with 10 ml af

dH20.

. ~ , , t t , ~, , ~ , ~ ~

! f ~\ ~.

~ ~ t t ~ ...... ! ~ ""'" ~ r 1

1 r f

~ ~ ~

31

The filters were removed, transferred to vials and dried

using infra-red lamps. After a sufficient period of cooling,

the filters were solubilized by the addition of 10 ml Aquasol . to each vials. The radioactivity of the filters was deter-

mined using a Nuclear-Chicago-Isocap/300 liquid scintillation

spectrophotometer. The samples were counted for 10 min at

0.25% 2 sigma level with a 14C program.

SEPARATION AND CHARACTERISATION OF THE OUTER-MEMBRANE

Separation of the outer-membrane (sucrose gradient)

A method was developed by Hancock and Nikaido (1978) for

separating the membranes of P. aeruginosa. Late exponential

cells were grown in 3 l of nutrient broth medium and then

harvested by centrifugation at 13 000 x 9 for 15 min. AlI

manipulations were performed at 4 C. The pellet was washed

once in 30 ml of 30 mM Tris-HCI pH 8.0. The washed pellet was

then resuspended in 15 ml 20% (w/v) sucrose in Tris-HCl 30 mM

pH 8.0 to which about 1 mg of Dnase and RNase was added. The

cell suspension was then passed twice through an ice cold

French Pressure c ~ll at 15 000 psi. One and a half ml of

lysozyme (1 mg/ml) was added and incubated for 10 min at room

temperatuxe. Whole cells and cell debris were removed by

centrifugation at 1 000 x g for 10 min. The supernatant was

diluted to 36 ml with 30 mM Tris-HCI pH 8.0. Six ml was

layered enta a sucrose step gradient containing a bottom

layer of 1 ml of 70% (w/v) ultrapure sucrose and 5 ml of 15%

(

(

32

(w/v) ultrapure sucrose in Tris buffer. The tubes were then

centrifuged in a Beckman SW 41 rotor for 1 hour at 39 000

rpm. The bottom 2 ml of each gradient (3) were removed and

applied to a further step gradjent containing steps of 1 ml

70%, 3 ml 64%, 3 ml 58%, and 3 ml 52% sucrose in Tris-HCI pH

8.0. The tubes were then centrifuged again at 39 000 rpm in

the SW 41 overnight (14-16 hours). Using a Pasteur pipette

the four bands were removed. Eact band was diluted with water

and washed twice using a Ti 55.2 Beckman rotor at 45 000 rpm

for 30-60 min at 4 C. Each fraction was resuspended in a

small amount of water and stored at -22 C. The fourth band

(lowest band) contains almost exclusively the outer membrane

fraction, under these conditions (Hancock and Nikaido, 1978).

Estimation of prote in concentration

The Lowry r'lethod, as modified by Markwell et al. (1978) 1

was used to estimate protein concentration~ using bovine

serum albumin as a standard.

12% Slab sodium dodecyl sulfate polyacrylamide gel

electrophoresis (SOS-PAGE)

The method used was from Bio-Rad taken from their

PROTEAN II Slab gel manual. The method was originally devel­

oped by Lugtenberg et al. (1975). Electrophoresis was per­

formed on the outer membrane fraction (fourth band) and the

cytoplasmic membrane (first band).

• ~.

Vertical slab gel

The PROTEAN II Slab Cell from BIO-RAD was used to

electrophorese the outer membrane and the periplasmic prot­

eins, using a stacking and a separating gel of 4.0% and 12%

acrylamide/Bis respectively. The sepacating gel contained

20.0 ml of a stock solution of 29.2% (w/v) acrylamide/ 0.8%

N'N'-Bis-methylene-acrylamide, 0.5 ml of 10% SOS, 12.5 ml

1.5M Tris-Hel, pH B.B and 16.75 ml distilled water. The

polyrnerization was initiated when 250 ~l of 10% ammonium

persulfate and 25 ~l of TEMED were added to the monomer

solution. The stacking gel contained 1.3 ml of 30%

acrylamide/Bis, 100 ~l of 10% SOS, 2.5 ml of 0.5M Tris-hel,

pH 6.8, 6.1 ml of distilled water, 50 ~l of 10% ammonium

persulfate and 10 ~l of TEMED.

Electrode buffer

33

The 5X electrode buffer stock was composed of 45.0 g

Tris base, 216.0 9 glycine and 15.0 9 SOS in 3.0 l of double

distilled water, pH B.3. Three hundred ml of stock buffer was

diluted with 1200 ml of double distilled water. The butfer

was then poured into the apparatus ta ensure the flow of

current through the gel.

Digestion buffer

The digestion buffer was composed of 1.0 ml 0.5M Tris­

HCI, pH 6.8, 0.80 ml glycerol, 1.6 ml 10% SOS, 0.4 ml 2B-

{

(

(

34

mercaptoethanol, 0.2 ml 0.05% bromophenol blue and 4.0 ml

double distilled water. To this digestion buffer 0.1 M MgC1 2

was added. The samples were diluted at least 1:4 with the

digestion buffer, and heated at 95 C for 4 min. The samples

were loaded using a 50 ~l Hamilton syringe. The best band

resolution was accomplished when the concentration of the

sample was 40 ~g/20 ~l (sample/well).

Running conditions

The electrophoresis was done at a constant current of 15

mArnps, for approximately 16 h or until the dye front just

exits the bottom of the gel, for a single gel system.

Protein standards

The low molecular weight protein standards include

rabbit muscle phosphorylase B (97 400 Da), bovine serum

albumin (66 200 Da), Hen egg white ovalbumin (42,699 Da),

bovine carbonic anhydrase (31 000 Da), soybean trypsin

inhibitor (21 500 Da) and lysozyme (14 400 Da). The protein

standards were treated in the same manner as the samples. The

standard curve was produced on a 10glo of molecular weight

versus the relatlve mobility of each protein to the dye front

or the edge of the bottom of the gel.

Fixing and staining the proteins in the gel

The gel was fixed and stained simultaneously using a

1

35

solution of 500 mg Coomassie Brilliant Blue R-250 dye dis­

solved in 1 l of 10% glacial acetic acid:25% methanol:65%

double distilled water. The gel was put in the solution

overnight at 37 C in a shaking water bath. After the stain­

ing, the gel was destained in the same solution, less the

dye. The destain was done at 30 C in a shaking water bath for

about 6 h. A sponge was added on top of the gel during the

destain procedure.

Atomic absorption ( Mg+2 concentration)

The PYE UNICAM SP9 atomic absorption spectrophotometer

was used to determine the concentration of Mg+ 2 in the outer

membrane fraction (as described earlier) of the wild type

(PU21) and the resistant substrains (PU21/RC150 and

PU21/RC500). The 3UNX/Mg (gas Ne) lamp was used as the light

source. The absorbance was read at  = 285nm. The air tank

pressure was set at 30 Ibs/in 2 and the acetylene at 9

Ibs/in 2 • The gas flow in the machine was set from 28 to 38 mm

for the oxygen and 20 mm for the acetylene. The PYE UNICAM

SP9 atomic absorption spectrophotometer user manual was

followed.

The Mg+ 2 standard solution was used to construct a

standard curve (concentration of Mg+2 versus absorbance at

285nm). The concentrations of Mg+ 2 standard use to construct

this standard curve were: 0.5, 2.0, 4.0, 6.0, 8.0 and 10.0

~g/ml. The final concentration of Mg+ 2 in the outer membrane

r '"

36

fraction was calculated as J.l.g of M9+2 /fl9 of proteine

RESPIRATORY ACTIVITY OF WHOLE CELL SUSPENSION

preparation of whole cell suspension

Four hundred ml of cells werc harvested after the appro-

priate time (as indicated in Results) of growth in PPGAS. The

cells were washed 3 times in PBS buffer composed of : 100 mM

NaCI, 10 mM Rel, 25 mM Na2HP04f 5 mM MgS04·7H20. The cells

were concentrated at a cell density equal to 12 mg dry wt per

ml. An optical density of 1.0 was aS!:lln1ed to be equivalent to

400 mg dry wt per litre. P. aeruginosa ArCC 9027, prepared in

this manner, contained approximately 7.8 mg of protein per ml

for the wild type and about 8.6 mg of protein for the resis-

tant RC-150.

Substrates and eguipment

AlI substrates were prepared as 600 mM solutions in PBS

buffer. The final concentration within the oxygen electrode

was 10 mM (50 ~l of concentrated substrates was added to the

oxygen electrode).

Oxygen uptake was assayed polargraphically at 36 C using

a Rank Brothers oxygen electrode (Rank Bros., Bottisham,

Cambridge, U.K.), an electrode power unit (U.K.C.-163) and a

recorder (Fisher Recordall, Series 5000). A fresh Teflon

membrane was prepared each time respiration studies were

[

.! ~ l'

~ ~ ~

~

','

..... -

.. ...

37

perforrned and the recorder recalibrated each time. The oxygen

chamber was sealed by screwing down the electrode stopper so

that the meniscus of the buffer solution was half way up the

port channel. AlI additions to the electrode were

administered using a 50-100 ~l capacity Hamilton syringe.

Respiration assays

The oxygen electrode contained 2.9 ml PBS buffer. The

oxygen chamber was sealed and the buffer was allowed to

equilibrate for 5-10 min at 36 C. One hundred ~l of washed

cell suspension were introduced into the chamber. Endogenous

oxygen consumption was measured for about 3 min after which

50 ~l of suhstrates were added and oxygen consurnption was

monitored for a further 5 min. The results were corrected for

endogenous respiration and expressed as ng ions 0 consumed

per min per mg proteine The mean value for the solubility of

oxygen in this medium was assumed to be 0.399 ~g atoms 0 per

ml (Chappell, 1964) .

(

RZSULTS

Ingram and Hassan (1975) found that when P. aeruginosa

ATCC 9027 was grown in the presence of chloramphenicol, an

outgrowth of resistant cells occurred after a relatively

brief period of growth inhibition. The growth and physiolo-

gical characteristics of the resistant strains of P.

aeruginosa were demonstrated and discussed by Irvin (1983)

and Mahmourides (1983). Consequently, resistance to

chloramphenicol in P. aeruginosa has generally been inter-

preted in terms of a "permeability" change, the nature of

38

which remains to be defined. This study will attempt to

demonstrate effects of chloramphenicol on the outer membrane

of P. aeruginosa.

EFFECTS OF CHLORAMPHENICOL ON THE OUTER MEMBRANE

5pheroplast formation experiment

Since chloramphenicol does not enter the cells (as

discussed previously), chloramphenicol must have some effect

on the outer membrane of P. aeruginosa. An experiment was

developed in order to examine if chloramphenicol has any

effect on the outer membrane of P. aeruginosa. If

chloramphenicol disrupted the outer membrane enough, then

lysozyme could enter the periplasmic space and act on the

peptidoglycan to form spheroplasts. 50 spheroplast formation

was initiated by treating the cells, wild type (9027 and

~ t

t' t (

~ ~ r, i ~

r ~

" . " ,

,~.

PU21) and resistant strains, first with chloramphenicol and

then with lysozyme.

39

A wide range of chloramphenicol concentrations was used,

starting from 10 ~g/ml up to 500 ~g/ml. After many trials, no

spheroplasts were observed under phase-contrast microscopy.

Manipulations of the temperature and incubation time did not

influence the outcome of the experiment.

When P. aeruginosa is treated with EDTA and lysozyme,

the cells either lyse or form osmotically fragile rods termed

osmoplasts (Eagon and Carson, 1965). 50 if the action of

chloramphenicol was similar ta that of EDTA, as a chelator,

it might be impossible or almost impossible to get

spheroplasts without lysing the cells. Another assay was

developed in order to get around this osmotic fragility of P.

aeruginosa. Instead of treating the cells with lysozyme,

penicillin G was used.

penicillin G experiment

P. aeruginosa is highly resistant to penicillin G. This

resistance is attributed to the relative impermeability of

the cell wall (Richmonds and Sykes, 1973 ; Richmonds and

Curtis, 1974 ; Brown, 1975). But if chloramphenicol could

disrupt the outer membrane, then penic~_lin G could enter the

periplasmic space and inhibit peptidoglycan synthesis. In

this experiment cells were treated (or not) with

chloramphenicol at concentrations of 150 and 500 ~g/ml for 30

(

(

40

min and then incubated with (or without) 25 or 100 ~g/ml of

penicillin G. The growth of the cells was monitored spectro­

photometrically at  = 660nm.

Figure 5 represents the wild type (PU21) treated for 30

min with 150 ~g/ml of chloramphenicol. A 1% inoculum was then

transferred to nutrient broth containing 25 ~g/ml of peni-

cillin G. The first two growth curves are identical and

demonstrate that when P. aeruginosa is not treated with

chloramphenicol, the cells are highly resistant to 25 ~g/ml

of penicillin G. But when the cells are treated with

chloramphenicol there is a decrease in the growth rate of the

cells as shown by the third curve. Incubation with penicillin

G still has no effect on the growth rate of the cells, as

shown by the last curve. So penicillin G is still excluded

from the periplasmic space by the impermeability barrier of

the outer membrane even after the cells have been treated

with 150 ~g/ml of chloramphenicol.

The same experiment was repeated with the resistant

substrain RC150/PU21 (Fig. 6). The growth rates of the

resistant cells were not affected by chloramphenicol,

penicillin G or the combination of both antibiotics. This

again proves that RC150/PU21 is resistant to chloramphenicol

and penicillin Gand that the action of chloramphenicol on

the outer membrane is insufficient to allow penicillin G to

penetrate the cells.

• ..

Figure 5

41

Fig. 5. Growth of PU21 in nutrient broth (exposed to 150 ~g/ml chloramphenicol and 25 ~g/ml penicillin G).

The cells were treated (or not) for 30 min with 150 ~g/ml of chloramphenicol (CM) and incubated with (or without) 25 ~g/ml of penicillin G.

Symbols: -- minus CM, minus penicillin G -&- minus CM, 25 ~g/ml penicillin G -. 150 ~9/ml CM, minus penicillin G

~ 150 ~9/ml CM, 25 ~9/ml penicillin G

t

-E c: o «D «D -w U Z C III Œ o CO III C

10

O.01~--~--~--~--~----L---~---L---J

o 1 2 3 4 5 TIME (hour.)

8 7 8

.....

t

42

Figure (

.

Fig. 6. Growth of RC150 in nutrient broth (exposed to 150

~9/ml chloramphenicol and 25 ~9/ml penicillin G) •

The cells were treated (or not) for 30 min with 150 ~q/ml of chloramphenicol (CM) and incubated with (or without) 25 ~q/ml of penicillin G.

Symbols: - minus CM, minus penicillin G -8- minus CM, 25 ~9/ml penicillin G ..... 150 ~g/ml CM, minus penicillin G ~ 150 ~g/ml CM, 25 ~q/ml penicillin G

-

--

.. ...

r 1

10

-E c 0 G) G)

."... -...... w (,) z < ID CI: 0 fi) ID <

O.01~--~--~----~--~--~--~----~--~

o 1 2 3 4 5 8 7 8 TI ME (houra)

(

43

In order to increase the action of chloramphenicol and

penicillin G, both concentrations were increased to 500 ~g/ml

and 100 ~g/ml respectively. Once again we can see (Fig. 7)

that penicillin G alone, even at high concentration, has

little or no effect on the growth rate of PU21 (curve 1 and

2) . But when the cells are treated with 500 ~g/ml of

chloramphenicol (curve 3), the growth rate is even slower

than before (Fig. 5). A 16 h lag phase is observed (curve 4)

when the cells are exposed to both antibiotics. After the lag

period, the growth rate of the resistant outgrowth is even

faster than when chloramphenicol is used alone.

The same treatment as above, was applied to RC-500/PU21

and was used as a control (Fig. 8). This substrain is highly

resistant to 500 ~g/ml of chloramphenicol, 100 ~g/ml of

penicillin G or the combination of both antibiotics. This

resistance is demonstrated by the growth curves of Fig. 8.

The curves are nearly identical and are not affected when

antibiotics are added to the medium.

The foregoing results were not conclusive. An experiment

was developed to try to assess the damage (OL change)

inflicted to the outer membrane by chloramphenicol.

44

.,.... Figure 7

Fig. 7. Growth of PU21 in nutrient broth (exposed to 500 ~g/mi chloramphenicol and 100 ~g/mi penicillin G) .

The ceIIs were treated (or not) for 30 min with 500 ~g/ml of chloramphenicol (CM) and incubated with (or without) 100 ~g/ml of penicillin G.

Symbols: - minus CM, minus penicillin G ~ minus CM, 100 ~g/mi penicillin G -fi- 500 ~g/ml CM, minus penicillin G ~ 500 ~g/ml CM, 100 ~g/ml penicillin G

-E c o CD CD -w (,) Z C III II: o CI) III <

10

O.Ol~--~----~--~----~--~----~--~

o 8 12 16 20 24 28

TIME (hours)

45

Figure 8

Fig. 8. Growth of RC500 in nutrient broth (exposed to 500

~g/ml chloramphenicol and 100 ~g/ml penicillin G) •

The cells were treated (or not) for 30 min with

500 ~g/ml of chloramphenicol (CM) and incubated with

(or without) 100 J,lg/ml of penicillin G.

Symbols: - minus CM, minus penicillin G

-I!r- minus CM, 100 ~g/ml penicillin G

--- 500 J,lg/ml CM, minus penicillin G

-e- 500 J,lg/ml CM, 100 J,lg/ml penicillin G

t

10

-E c 0 CD CD ....... -

-<V. W U Z < ID a: 0 CI) ID <

O.01~--~---L--~----~--J----L--~~~

o 1 2 3 4 5 8 7 8 TIME (hour.)

The release of alkaline phosphatase

Cheng et al. (1970b) have shown that in P. aeruginosa,

alkaline phosphatase is located in the periplasmic space.

Most agents which disrupt the outer membrane also cause the

release of this enzyme. Late log cells (14 h) were washed

with different solutions, as shown in Fig. 9, in order to

release alkaline phosphatase in the medium. The washes were

analysed by a sensitive colorimetrie method involving pNPP.

46

The highest activity, 0.2 units/mg protein, was observed in

the culture filtrate before any washing. When the cells were

washed with 0.2 M Mg 2+, high alkaline phosphatase activity

of 0.13 units/mg protein was also observed. No, or very Iow,

activity was detected when the cells were washed with differ-

ent concentrations of chloramphenicol. Only an activity of

0.04 to 0.05 units/mg protein was measured. Alkaline

phosphatase is not released into solution when the cells are

exposed to chloramphenicol. Again it is observed that the

effect of chloramphenicol is insufficient to disrupt the

outer membrane and cause alkaline phosphatase to leak out of

the periplasmic space.

47

-,

Figure 9

l

.

Fig. 9. Alkaline phosphatase (APase) activity in 9027.

Cells were washed harvested and washed after 14 h

of growth in PPGAS giving O.D. (660nm) of 1.3.

Washing solutions used to release alkaline phosphatase.

A: Culture filtrate B: 0.2M Mg2+ in O. 01M Tris pH 8.4 C: o . 2M Mg2+ in dH20 (pH 8.4)

D: 150 J,lg/ml chloramphenicol (dH2O)

E: 500 J,lg/ml chloramphenicol (dH2O)

F: 150 J,lg/ml chloramphenicol in 0.01M Tris pH 8.4

G: 500 J,lg/ml chloramphenicol in 0.01M Tris pH 8.4 H: dH20

I: 0.01M Tris pH 8.4

.......

~

-

.....

, ,

--

• ..

A

B

C

0

E

F

G

H

o

0.2

0.06 0.1 0.16 0.2 0.25

APas. activity (unite/mg protein)

(

(

(

48

The binding of chloramphenicol to P. aeruginosa

As reported by Vasquez, 1963 ; Gaffney, 1981 ; Irvin,

1983 , it was not possible to demonstrate the intracellular

accumulation of chloramphenicol, even in sensitive cells.

Since resistance cannot be ascribed to drug detoxification,

exclusion is the alternative mechanism to considere Attempts

were made to measure the binding of 14C-chloramphenicol by

standard filtration assays. It was not possible to demon­

strate any binding of chloramphenicol to sensitive or resis­

tant cells. The values of 14C-chloramphenicol found on the

filters after the assays were insignificant and had no rela­

tionship to the external chloramphenicol concentration, cell

density or incubation time. The counts on the filters were

extremely similar regardless of the presence or absence of

cells, 14C-chloramphenicol was observed to bind to the fil­

terse The composition and temperature of wash and assay

buffers were modified without any success. Either the cells

do not bind 14C-chloramphenicol or the assay is not sensitive

enough so that the drug is dissociated from the cells during

the manipulations.

It was decided to examine the outer membrane in greater

detail. If chlo~amphenicol has any effect on the outer mem­

brane of P. aeruginosa the resistant cell membrane should

show sorne differences compared to the sensitive cells.

49

SEPARATION AND CHARACTERISATION OF THE OUTER MEMBRANE

The sucrose step gradient method developed by Hancock

and Nikaido (1978), as described in Materials and Methods,

was used in order to separate the inner and outer membranes

of P. aeruginosa (PU21). To separate the membrane fractions,

4 different sucrose concentrations were used (52, 58, 64 and

70%). With this sucrose step gradient, 4 distinct bands were

observed : the first band (on top), was composed of the

cytoplasmic membrane; the last band (bottom), was composed of

the outer membrane and the 2 middle bands were composed of a

mixture of both membranes. The 2 middle bands were not used

in this study.

These membrane fractions were used to compare the outer

membrane protein profiles and the outer membrane Mg2+ concen-

trations of the sensitive and resistant cells.

Outer membrane protein profiles

The outer membrane fractions of both resistant and

sensitive cells were electrophoresed on a SDS polyacrylamide

gel to separate the protein components. The gel is shown in

Fig. 10. An extra band with an apparent molecular weight of

49 x 103 Da was only observed in the resistant cells. From

Hancock (1990), this band may represent the outer membrane

prote in P which should have an apparent molecular wejght of

48 x 10 3 Da (Table 1), or it may represent a new protein not

yet observed. Two other bands, contrary to the first one,

-

50

Figure 10

.,

Fig. 10. Twelve percent SOS-PAGE of outer membrane (OM) from

chlorarnphenicol resistant and sensitive strains of

P. aeruginosa.

Lane A: Low molecular weight prote in standards.

Lane B: OM of sensitive PU21.

Lane C: OM of resi.stant strains RC150.

Lane D: OM of resistant strains RC500.

Lane E: Ouplicate of Lane B.

Lane F: Ouplicate of Lane C.

Lane G: Duplicate of Lane o. Lane H: Low rnolecular weight protein standards.

Numbers on the left indicate molecular weights (10 3) of

standard proteins.

Numbers on the right indicate rnolecular weights (103) of

bands of interest.

Letters indicate sorne major outer membrane proteins.

.~

~

.-

97.4 66.2

42.7

31

21.5

1 4.4

ABCDEFGH

..

------1

9 1

2 9

. t

r

\ .. were only observed in the sensitive celis. Their apparent

moiecular weights are 19 x 10 3 Da and approximately 10 x 103

51

Da. The smallest size band represents the outer membrane

protein I (OprI, Table 1). No evidence of a 19 x 10 3 Da outer

membrane proteiu as reported in the Iiterature was observed,

so that this outer membrane protein might be observed here

for the first time.

The 'luter membrane protein P (OprP) is present in celis

grown on lo~ phosphate medium (Hancock et al., 1982); it is

aiso involved in phosphate transport (Poole a~d Hancock,

1986). OprI is a low molecular weight lipoprotein and is

covaiently attached to a sIngle fatty acid residue (Mizuno

and Kageyama, 1979). Depending on the strains, OprI may be

covalently or non-covalently attached to peptidoglycan. It is

involved in the structure of the outer membrane. The new band

at 19 x 10 3 Da is within the range of the lipoproteins and is

also probably involved in the mechanical structure of the

outer membrane of the sensitive cells. Proteins Dl (OprB), E

(OprE), F (OprF) and H2 (OprL) were aiso observed in aIl 3

strains. They do not seem to be involved in the resistance

mechanism of P. aeruginosa since no differences were observed

between resistant and sensitive cells.

From these differences we can spe< .. ate that there may

be sorne structural differences in the outer membrane of

resistant celis compared to sensitive cells. These structural

modifications may have lowered the phosphate level 50 that

.'

',~

prote in P was produced in order to counteract these changes

in the outer membrane.

Mg 2+ concentration in the outer membrane

52

The Mg2+ concentration of each outer membrane fraction

was measured using an atomic absorption spectrophotometer.

The concentration of Mg2+ in the resistant cells was doubled

compared to the sensitive cells (Fig. 11). The concentration

averages are 3,0 x 10-3; 6,0 x 10-3 and 5,6 x 10-3 Jlg Mg21/Jlg of

outer membrane proteins for the wild type (PU21), RC-IS0 and

RC-SOO respectively.

The resistant strains are mOdified, most likely in the

envelope, as shown previously by the protein profiles, such

that the interaction with divalent cations probably results

in a significantly more effective barrier against

chloramphenicol. It was shown that when the resistant cells

are grown in a law Mg2+ environment, the resistant cells

revert back to sensitive (Irvin, 1982). ThiF "restructuring"

of the outer membrane appears ta involve an increase in

divalent cations as demonstrated by the higher Mg2+ concen­

tration in resistant cells.

'.

53

Figure 11

Fig. Il. Mg+ 2 concentration in the outer membrane of PU21,

RC-1S0 and RC-SOO.

Mg2+ concentration = Mg of Mg2+

~g of outer membrane prote in

• ..

c: o -

8X10-3~--------------------------------~

~ 4X10· 3 -c ., CI C o CI

o Wlld Type (PU21) RC-150 RC-500

(

{

54

RESPIRATION ACTIVITY OF WHOLE CELL SUSPENSION

A physiological study of the cells was conducted by

observing the respiration rates of 9027 and RC-150/9027.

These strains were tested in the presence, or absence, of

chloramphenicol using 4 different substrates : glucose,

gluconate, succinate and alanine. As su~narized in Table 2,

we can observe a general decrease in respiration when

chloramphenicol is added, but this decrease is not

significant, since a larger difference is seen at different

growth cycles of the cells. This reduction of respiration

rate seen in cells tested in the presence of chloramphenicol

may be due to a decrease in transport of substrates. Irvin

and Ingram (1980) have already shown that the resistant cells

are in fact defective in amino acid transport, so the

decrease in respiration rate observed here may only be due to

a secondary effect of the defective transport system.

Table 2. Respirationa in 9027 and RC150/9027.

9027 b RC150/9027 b

Substrate - CM + CM - CM

Earl~ log cells

Endogenous 58 ND 64

Glucose 6 ND S2

Gluconate 34 ND 66

Succinate 108 ND 136

Alanine 129 ND 61

Mid-log cells

Endogenous 115 146 61

Glucose 86 36 40

Gluconate 46 87 36

Succinate 72 55 139

Alanine 121 80 61

Late log: cells

Endogenous 47 57 87

Glucose 10 10 74

Gluconate 302 249 221

Succinate 0 0 101

Alanine 60 37 97

AlI respiration rates were corrected for endogenous respiration.

+ CM

ND

ND

ND

ND

ND

72

35

32

117

46

95

54

166

88

78

55

a Rates of oxygen consumption are expressed in nanogram atorns oxygen per minute per mg cell proteine

b Cells grown plus and minus chloramphenicol (CM) at 150 J1g!ml.

(

(

56

DISCUSSION

There is the possibility of 3 major mechanisms ~f resis­

tance of P. aeruginosa against chloramphenicol. The drug can

be acetylated and inactivated by CAT enzymes (Shaw, 1967;

Shaw and Brodsky, 1968), ribosomes can be altered 50 that

they do not bind chloramphenicol (Osawa et al., 1973) or the

cells can become impermeable to the drug (Bllrns et al.,1989;

Chamberland et al., 1989; Hirai et al., 1987). Detoxification

by acetylation is the predominant mechanism of resistance

against chIoramphenicol in both gram-positive and gram-nega­

tive bacteria (Shaw, 1974; Fitton et al., 1978). However, the

Ievel of acetylating activity in P. aeruginosa is barely

detectable and is insufficient to explain the high level of

resistance observed in this organism (Okamoto et al., 1967;

Sompolinsky et al., 1968). Irvin (1983) also demonstrated

that the strains used in this study (RC150 and RCSOO) produce

no or barely detectable acetylating activity. Ribosomal

alterations were found in Bacillus species (Osawa et al.,

1973) but this mechanism was never observed in gram-negative

bacteria. The only mechanism of resistance of P. aeruginosa

against chloramphenicol remaining, is, impermeability.

Initially it was thought that chloramphenicol altered

the outer membrane of P. aeruginosa which could explain the

Iag period observed when exposing the sensitive cells to high

Ievels of chIoramphenicol. The cells, to protect themselves,

l

57

would modify their outer mem~rane so as to render it imperme­

able to the drug. This hypothesis was tested by 3 different

expertments. In the first, spheroplast formation was tested

by treating the cells (sensitive and resistant) with the drug

and then with lysozyme. After many trials, using different

concentrations, incubation times and media composition, no

spheroplasts could be observed. The second experiment con­

sisted of exposing the sensitive cells to chloramphenicol and

then to penicillin G. As aIready mentioned, P. aeruginosa is

resistant to penicillin Gand this resistance is attributed

to the relative impermeability of the cell wall (Richmonds

and Sykes, 1973; Richmonds and Curtis, 1974; Brown, 1975).

Using the above hypothesis, chloramphenicol could disrupt the

outer membrane, permit penicillin G to enter the periplasmic

space and kill the susceptible cells. Only a low synergistic

effect was observed when the cells were exposed to 500 ~g/ml

of chloramphenicol and then 100 ~g/ml of penicillin G (Fig.

7). The results were not conclusive so a third experiment was

developed. The effect of chloramphenicol on the outer

membrane of P. aeruginosa was monitored by measuring the

amount of alkaline phosphatase released by the sensitive

cells when exposed to high concentrations of chloramphenicol.

Figure 9 clearly shows that no (or very low) alkaline

phosphatase activity could be observed when the cells were

exposed to chloramphenicol. The previous hypothesis of

chloramphenicol attacking the outer membrane of P. aeruginosa

.,. ,(

58

was discarded after the inconelusive results of these experi-

ments.

The next logical step was to take a eloser look at the

outer membrane of P. aeruginosa and determine whether there

is any difference in the composition of the outer membrane of

sensitive versus resistant eells. Figure 10 demonstrates the

outer membrane protein profiles of both sensitive (PU21) and

resistant cells (RC150 and RC500). Three major differences

can be seen between wild type and resistant strains. One

extra-band of 49 000 Da was observed in the resistant

strains. Two extra-bands of 19 000 Da and about 10 000 Da

eould only be seen in the wild type strains. The 49 000 Da

band ean either be a new band or Opr P which has an apparent

monomer moleeular weight of 48 000. Opr P is involved in the

transport of phosphate and it is an important component of

the high-affinity phosphate-specifie transport (Pst) system

(Poole and Hancock, 1986a). The simplest explanation could be

that Opr P may have been indueed in order to help aceumulate

phosphate for the "re-structuring" of the outer membrane in

the resistant cells. Opr I, on the other hand, is not present

in the resistant strains. The lipoprotein Opr l is involved

in the structural integrity of the cell wall and its loss may

be interpr.eted as a primary or secondary effect from the

alterations in the outer membrane. The new 19 000 Da band

observed in the sensitive cells is within the ra~~e of the

lipoproteins and may be, like Opr I, involved in the

59

physical structure of the outer membrane. Ali of these

hypotheses are purely speculative and it is not known if

these changes in the outer membrane protein profiles are due

to primary or secondary reactions from the "re-structuring"

of the outer membrane. It is, however, quite clear that these

observations demonstrate that there is sorne sort of physical

modification or alteration in the outer membrane of resistant

cells of P. aeruginosa against chloramphenicol. Other

researchers observed differences in the outer membrane pl.'O-

te in profiles of Pseudomonas species against different type

of drugs. Hirai eë al. (1987) observed a new 54 kDa outer

membrane protein in a norfloxacin resistant P. aeruginosa.

They suggested that this new prote in may block norfloxacin

uptake by an unknown mechanism, since no other changes were

observed and that the resistant cells were impermeable to the

drug. Chamberland et al. (1989) found that the resistance of

P. aeruginosa to quinolone was correlated with the reduction

of outer membrane protein G and the loss of a 40 kDa protein.

This quinolone resistant P. aeruginosa was also cross-resis-

tant to chloramphenicol. They once again suggested

impermeability as the mechanism of resistance.

Divalent cations, especially Ca2+ and Mg2t, are aLl

important component of the outer membrane of P. aeruginosa.

They are involved in the structure, stability and function of

the cell envelope of grarn-negative bacteria (Cheng et al",

1970a; Stan-Lotter et al., 1979; Brass et al., 1981; Hancock,

60

1984). In P. aeruginosa, the presence of polymyxin B, EDTA

and other divalent cation specifie chelacors increases the

susceptibility to antimicrobial agents (Hancock, 1984). lrvin

and Ingram (1982) ~'1owed that when the Mg2+ concentration in

the medium was lowered below 1mM, the resistant strains

became susceptible to chloramphenicol. This infl~ence of

divalent cation was immediate and is not a result of pro­

longed growth in divalent cation depleted medium. Irvin and

Ingram (1982) speculated that the resistant strains may be

altered in the cell envelope such that the diva lent cations

played an important role in the structure of the outer mem­

brane as a more effective barrier against chlor~mphenicol and

that this resistance involves an increased diva lent cation

requirement. Figure Il demonstrates that thn 'esistant

strains, RC150 and RC500, had twice as much Mg2t in the

envelope of the resistant strains and this is consistent with

the speculation of Irvin and Ingram (1982) that the resis­

tance rnechanism involves an increased divalent cation

requirement in order to "re-organize" a more effective outer

membrane against chloramphenicol.

Many researchers (Irvin, 1983; Gaffney et al., 1981;

Vasquez, 1963) have tried for many years to measure the

uptake of chloramphenicol inte cells of P. aeruginosa. No one

was able te directly measure the uptake of chloramphenicol

either by filtration or rapid centrifugation techniques. The

binding of chloramphenicol to the cells of P. aeruginosa

ft

61

u=ing standard filtration transport assay was conducted in

this study. No binding of chloramphenicol to the sensitive or

resistant cells of P. aeruginosa was observed. More recently,

Abdel-Sayed (1987} using sensitive P. aeruginosa, observed

the depletion of chlorarnphenicol from the medium. They

assumed that the missing 14C-chloramphenicol was to be found

within the cells. They did not study the accumulation of

chloramphenicol within resista~t cells. On the other hand,

using P. cepacia, Burns et al. (1989) measured the depletion

of chloramphenicol from the medium. They observed a 10-fold

decrease in intracellular accumulation of chloramphenicol

within the resistant cells and concluded thdt the rnechanism

of resistance of P. cepacia against chlt)ramphenicol was

impermeability. Irvin and Ingram (1980) demonstrated that the

resistant strains used in this study had a reduced amine acid

uptake compared to the wild type PU21. They established that

this amino acid uptake reduction was due to the

impermeability barrier of the outer membrane of the

chloramphenicol-resistant strains.

AlI the evidence thus far suggests that the resistance

mechanism of P. aeruginosa against chloramphenicol involves

decreased permeability. This permeability barrier can occur

either at the outer membrane or the cytoplasrnic membrane

level. Active transport of chloramphenicol across the cyto-

plasmic membrane of Haemophilus influenzae has been observed

(Burns and Smith, 1987). This impermeability barrier is very

62

unlikely in P. aeruginosa since the resistant strains are

also cross-resistanr. to other drugs such as penicillin G,

quinolones and polymyxin B. In addition, aIl the obse~vations

made in this study such as differences in outer membrane

protein profiles and the Mg2+ content of the outer membrane

strongly suggest a "re-organization" of the outer cell wall

of P. aeru~nosa. This "re-organization" of the cell wall

would make it more impermeable to chloramphenicol.

Based on the fa ct that outer membrane prote in profiles

differences were observed and that Mg2 + concentrations in the

outer membrane varied, the LPS patterns should be studied.

This high level of resistance against chloramphenicol might

also involve changes in LPS composition, such that the higher

concentration of Mg2+ and the outer membrane p~otein

differences may aIl interact in order to make the envelope of

P. aeruginosa more impermeable to chloramphenicol.

..

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( .

=

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