`
1 33075~
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Thi9 invention relates to the use of lysostaphin -
~in the treatment and prevention of staphylococcal infection
and, in particular, to the treatment and prevention of ~.
staphylococcal bovine ma~titis. .
Lyso~taphin is a bacteriocin secreted by a ~ingle ; ;
known strain of Staphylococcus simulans originally isolated
and named Staphylococcus staphylolyticus by Schindler and ~ -
Schuhardt. The production of ly~ostaphin by S. sta~hylo
~yticus ha~ been described previou~ly in U.S. Patent No.
3,278,378 is~ued October 11, 1966 and ln Proceeding~ of the
Natlonal Academy of Science~, Vol. 51, pp. 414-421 ~1964). `~
The ~ingle organl~m S~_staphylolyticus ~NRRL B-2628) which
.. .. . .
produced ly~o~taphin wa~ recently identified as a biovar of .:
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26704-364/2622
1 33075~
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S. simulans by Sloan et al., Int. J. System. Bacteriol.,
Vol. 32, pp. 170-174 (1982~. Since the name
S. staphvlolyticus is not on the Approved List of ~acterial
Names, the organism producing lyso3taphin has been redesig-
nated as S. ~imulan~.
~ acteriocins are prote;ns secreted by bacteria
that kill and sometimes lyse related bacteria. For example,
lysostaphin lyse~ and kills practically all known staphy-
lococcal specie~ but iq inactive against bacteria of all
other genera. Lysostaphin, isolated from culture filtrate
of S, ~imulan~ ~NRRL B-2628) grown according to published
references, is an endopeptidase which cleave~ the polyglycine
cross-links of the peptidoglycan found in the cell walls
of staphylococci. In addition, cultures that produce ly50-
staphin appear to be resistant to its activities while cul-
tures grown under non-lyso~taphin producing conditions are ~;
sen~itlve.
Previoua studies have shown that lysostaphin can be
produced by fermentation techniques wherein S. simulan~ is
grown in liquid culture. Such fermentation techniques are
described in U.S. Patent No. 3,278,378 i~sued October 11,
1966 and in Proceedlings of the National Academy of Science~
Vol. 51, pp. 414-421 ~1964). Various improvement~ in the
production of lysostaphin by fermenta~ion technique~ have
also been made as documented in U.S. Patent~ Nos. 3,398,056,
i~sued August 20, 1968, and 3r594~84~ issued July 20, 1971.
The latter two references disclose improvements to culture
medium and inoculation techniques whereby the production of
lysostaphin by fermentation can be accelerated and improved.
~ysostaphin i9 produced by S. ~lmulane during exponential
26704-364/2622
, :, ;
1 33075~ ~
growth as an inactive precursor. The proenzyme is converted
to active mature enzyme by protease produced by stationary
phase cultures of S. simulans.
In addition, ly~o~taphin can be produced by recom-
5 binant microorganisms, including strains of E coli, Bacillus -~
subtilis and B. sphaericus which express the lysostaphin
gene. In contrast to the natural production, lysostaphin
accumulates during exponential growth in the culture medium
of recombinant lysostaphin producing strains as fully pro-
cessed mature active enzyme and is free of ~taphylococcal
immunogenic contaminants. -~
Bovine ma3titis i~ a costly problem to the dairy ~ -
industry, costing over $2 billion per year in the United
State~ alone. The disea~e is e~timated tQ affect 50 per cent -~
of American dairy cows to some degree, leading to unusable
milk, decreased milk production, and, in cases of severe
infection, the death of the animal.
Ma~titis i~ cau~ed by infection of the milk gland~, ` ;-~
prlncipally by Sta~hYl~coçcus 3~ or St~eptococcus
~9~1~9~L~, and to a les~er degree by E._coli and other
gram-negatlve bacteria or combinations thereof. Most
streptococcal infection~ have proven to be ef-fectively treat~
able using conventional antibiotic therapy. Staphylococcal `;
mastitis has, however, proven more difficult to cure.
Traditional prevention of bovine mastitis can
involve a complex regimen Oe daily teat-dipping with a
disinectant solution, ~See, J. S. McDonald, 6 Veterinar~ `
Clinlca of North America Larqe Animal Practice 269 ~l9841)
and ma~, in some case~, involve antibiotic-containing teat
dips. Routine antîbiotic therapy must be approached with
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26704-364/2622
~ 3 3
caution, however, to minimize selection for antibiotic resis-
tant strains. When infection does occur, intramammary infu-
sion of antibiotics i9 indicated. Antibiotic therapy of this
kind can reduce the infection so that the ~ilk produced is
saleable, but it generally does not lead to complete elimina-
tion of the causative organism.
In the past, staphylococcal mastitis has shown a
poor respon~e to antibiotic therapy and a tendency for infec-
tionR to recur and become chronic. Studies on mastitis have
indicated that part of the problem in treating mastitis is
that a significant number of staphylococci remain viable in ~ ~-
the mammary gland within phagocytic polymorphonuclear neutro-
phil leukocytes ~PMN). It is believed that the staphylococci
within the PMN are protected from the effects of the anti-
15 biotic, and, when lysis of the leukocyte occurs, the phago- `~
cytized staphylococci may provide a renewed source of ;;
mastitis-produclng ~taphylococcal regrowth.
Studles on the possible mechanism of antibiotic
evasion o~ ph~gocytized staphylococci in mastitis treatment
20 3how that lyso~taphin had been re~ected as a candidate for ~ ~-
destroying phagocyti~ed staphylococci. Craven et al., 29
~esea~ch in Veterinar~ ience 57 (1980); Craven et al., 21
Antimicrobial Aqents and Chemotherapy 618 ~1982); Craven
et al., S Comp. Immiun. Microbial. Infect. Dis. 447 (19~2))
~-~ 25 Craven et al., 51 Journal o~ Dairy_~esearch 513 (1984).
In these experiment lysostaphin was used n vitro as a
pretreatment to destroy extracellular staphylococci prior
to exposing the phagocytized ~taphylococci to cloxacillin,
gentamicin or lyso~taphin. Craven et al.'s results strongly
suggest that lysostaphin would have no effect on mastiti~
'- , .,,.,~,
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26704-364/2622
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since intracellular staphylococci were still viable after 20
hours of incubation in a lysostaphin containing solution. 51
Journal of Dairy Research at 515-516, and Table 2.
Lysostaphin has also been reported to penetrate
human monocytes. Since monocytes are a different cell type
than PMNs, this human model is not likely to be applicable to
the treatment of bovine mastitis (van den 8roek et al., 21
Scand. J. Immunol 189 (1985))
Lysostaphin has also been shown to be effective in
the treatment of staphylococcal renal absce~ses in mice,
particularly when u~ed in sequence with the administration of
methicillin. Dixon et al., 41 Yale J. Biol~ Med. 62 (1968).
In man lysostaphin has also been used a~ a thera-
peutic agent for treatment of chronic nasal staphylococcal `
infections (Quickel, Jr. et al., 22 A~Plied MicrobioloqY 446
~1971)). In one case of a resistant staphylococcal infec- -
tlon, lysostaphin was given 3y~temically (Stark et al., 291
Mediçal Intelllqence 239 (1974)). In general, however, there
has been great ~kepticism and reluctance in the medical and
20 veterinary communitles concerning the systemic administration
o~ lysostaphin. Lysostaphln was considered to be too highly ;~
immunogenia to have general u`e for anything but topical ~ ~;
application~.
SUMMARY OF THE IN~7ENTIO~
i: i~ .~, ....
~ 25 It has now been found that lysostaphin can be used ;~ ~
,. :
with surprising effectiveness to prevent and/or cure staphy-
lococcal mastiti~, even in its chronic form, without any
adverse immunogenic effeat~. A~ a prophylactic, ly~ostaphin ~ -
can be introduced as part of a daily teat-dipping regimen.
;' ~ " '
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1 3307 5~
Lysostaphin can be used alone but preferably, the teat-dip
will include lysostaphin; other bacteriolytic agent~ such as
mutanolysin, a bacteriocin produced by Stre~tococcus
qlobisporus which is effective against streptococci: and ~ -
lysozyme, a muralytic enzyme which hydrolyzes the
polysaccharide backbone of the peptidoglycan in the cell
walls of Gram positive and Gram negative bacteria. The
formulation may also contain a chelating agent such a~
ethylenediamine tetraacetate (EDTA); and a mild surfactant
which has been found to potentiate the killing of the
bacteria. Suitable mild surfactant~ include, inter alia, --
esters of polyoxyethylene sorbitan and fatty acids (Tween
series), octylphenoxy polyethoxy ethanol ~Triton-X series),
n-octyl-B-D-glucopyrano~ide~ n-octyl-B-D-thioglucopyranoside
n-Decyl-3-D-glucopyranoside, n-Dodecyl-~-D-g}ucopyranoside,
and biologically occurring ~urfactants, e.g., fatty acids,
glycerides, monoglycerides, deoxycholate and esters of
deoxycholate.
In addition to the prophylactic use of the broad-
spectrum teat dip, various components of the teat dip can
be in~u~ed into the infected udder to eliminate the bacteria ~ ~
and cure ma~titi~, e.g., ly~ostaphin alone or with a mild ~ ;
surfactant which surprisingly potentiates the staphylocidal ~`
effect of lysostaphin more than 1000 times. Furthermore,
the combination of lysostaphin and penicillin also exhibit~
synergy such that a lOOO fold increase in the killing of ;~
staphylococci i`s observed in vitro. Therefore, a formulation ~ -
for therapeutic infusion can also include penicillin, or a
mild surfactant, with or without a chelating agent. ~
: ' ,
! 6
133075'~
Infusion3 of a therapeutically effective amount
of lyso~thaphin, with or without surfactant, EDTA,
penicillln or other potentiating agents, are used to
achieve elimination of the staphylococcal infection.
Preferably such infusions contain between 2 and 400 mg
lysosthaphin when no potentiatin~ agents are present. In
combinations containin~ potentiating agents, the required
effective doses of lysostaphin can be lowered (as a result
of it~ ~ynergistically enhanced activity) by as much as
1000-Pold.
Synergistic bactericidal activity of lysostaphin
and penicillin was observed even upon administration to
penicillina~e-positive 5. aureus and methicillin-resistant
S. aureus ("MRSA" ) . MRSA are usually resistant to
multiple antibiotics and are particularly problematic,
especially in humans, as well as difficult to kill. The
lysostaphin/penicillin comblnation would be indicated for
use in speciflc situations where grave MRSA infection
cannot be controlled by conventional antibiotic (e.g.
penicillin? therapy. In addition, penicillin and other
similar actin~ aubstances may also be useful together with
lyoostaphin as an agent a~ainst ~taphylococcal infection
and contamination.
While the utility of the lysostaphin containin~
28 ~ormulations according to the invention is illustrated
using mastitis treatment, the enhanced effectiveness of
the lysostaphin in thesq formulations makes them suitable
~or a number of other applications involvin~
staphylococcal in~ectlon and contam~nation. Thus, the
formulations could be used to control 6taphylococcal
in~ections by incorporatin~ them into wound dressin~s and
medication~, disinfectant crubs, wipes or lotions, or in
sur~ical implants. The formulations might al~o be used
~or cleaning of medical instruments, and of floors, walls,
bedding and the like in c1rcumstances where environmental
disinfection is desired. Other potential uses include use
as a nasal infusion to reduce intra-nasal carriage of
staphylococci, and food related uses such as treatment of
1 33075~
7a
meat, eggs, cheese and fish or food packaging and handling
equipment.
According to one aspect of the present invention,
there is provided a composition for killing staphylococci
comprising lysostaphin and at least one agent which
synergistically enhances bactericidal activity of
lysostaphin selected from the group consisting of
penicillin, synthetic penicillins, other cell wall-active
antibiotics, chelating agents, mild surfactants and other
membrane active agents in amounts effective to kill
staphylococci.
According to another aspect of the present
invention, there is provided the use of a therapeutic agent
comprising lysostaphin in an acceptable carrier for
treating staphylococcal mastitis by intramammory infusion.
According to a further aspect of the present
invention, there is provided the use of a teat dip
comprising ~rom about 0.01 to 10.0 ~g/ml lysostaphin in a
suitable carrier to prevent bovine mastitis.
In the accompanying drawing, Figure 1 shows a
chromatogram of lysostaphin produced by transformant B.
sphaericus strain 00 containing the recombinant plasmid
p~C16-lL whioh codes for lysostaphin.
;
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1 33075~
DETAILED DESCRIPTION OF THE INVENTION
Lysostaphin for use according to the claimed
invention can be obtained from either natural or
recombinant sources. Preferably, the lysostaphin is
obtained from Bacillus sphaericus strain 00 containing a
recombinant plasmid which directs the synthesis o~
lysostaphin, as this provides for both high levels of
lysostaphin production substantially free from
staphylococcal immunogenic contaminants and facile
lysostaphin purification since the lysostaphin accumulates
directly in the growth medium. Bacillus sphaericus
tranformants containing the plasmid pBC16-lL have been
found to be particularly suited for this purpose, although
other strains are also useful as a source of lysostaphin.
One method Por obtaining lysostaphin from micro-organisms
transformed by recombinant plasmids containing the gene
which codes for lysostaphin is fully disclosed in U.S.
Patent 4,931,390 (Recsei).
Method of Treatment
Prophylactic treatments for bovine mastitis
according to the invention involve the use of lysostaphin-
containing teat dips. Lysostaphin-containing teat dips
provide effeative prevention of bovine mastitis when used
before and after every milking. Pre~erably, the
preventative regimen is used for all cows in the herd. The
teat dips comprise about 1.0 ~g/ml lysostaphin in an
acceptable carrier. In addition, teat dips for use
according to the invention may include about 1.0 ~g/ml
mutanolysin, about 10 ~g/ml lysozyme, and a mild
surfactant. Acceptable
~ 26704-364/2622
1 33075~
carriers are those which provide a buffered medium of approx-
imately pH 8.0 and include aqueou~ buffers or hydrophilic
ointment base For example non-ionic detergents, fatty
acids or other mild surfactants, protein carriers, such as
serum albumin or gelatin, powdered cellulose and carmel can
be used as a carrier. The teat dip according to the inven-
tion may also advantageously include chelating agents, such
ag EDTA, colorants, and humectants, such as glycerol or
~orbitol.
Mutanolysin is obtained from Streptomyces qlobi~-
~orus. Lysozyme is obtained from chicken egg whites.
Intramammary infusion of lyso~taphin can be used to ~ ~;~
effectively treat infected animals who have developed either `~
chronic or acute ~taphylococcal bovine mastiti~ despite
lS prophylactic treatment. A single dose of from 2 to 400 mg
lysostaphin per milk gland will eliminate the infection and
cure staphylococcal maYtitis in mo~t instance3. Additional
doses of lysostaphln may be indicated where the inection is
persistent. Do~es ~lignificantly higher than 400 mg are not
Z0 recommended a~ they can lead to unwanted and potentially
adverse ~ide effects including transient swelling, tender~
ness, and reduced milk production. These effects are limite`d
to the treated gland, however, 90 that higher doses to a ;;
single gland may be appropriate in severe and life-
threatening situations. In life-threatening cases, the route
of admini~tration could also include ~ites other than the
infected gland ~o as to achieve systemic delivery, i.e.,
intravenous, subcutaneou~, or intramuscular, and rectal or
oral administration of suitably encapsulated formulations in ;~
,' ' .
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~ 26704-364/2622
.
~` 1 33075~ ~i
which the lysostaphin i~ protected from inactivation in the
gut.
It has also been found that infusion of a combina-
tion of lysostaphin and penicillin is surprisingly much more
5 efficaciou~ than lysostaphin alone because of an apparent ~ ;~
¦ synergistically enhanced bactericidal activity of this
combination. In addition, it is believed that the thera- ~-
peutic lysostaphin formulation may also include other agents
which potentiate the bactericidal activity of lyso~taphin, ~ ;
lO for example, synthetic penicillins and other antibiotics, ;~
chelating agents, mild surfactant~, ~e~g., deoxycholate) and
other membrane active agents which may facilitate penetration
of lysostaphin to the site of infection. In formulations ;; ;~
that include e.g., penicillin, the dosage of lysostaphin can ;~`~
be decreased as a result of the potentiated bactericidal
activity of lysostaphin. Since too high a dose of lyso~-
taphin can induce unwanted and potentially adverse ~ide~
~, :
e~fects, this synergistic efEect is significant not only for
efflcacy but also for avoidance of potential side effects.
Examples 1~4 ;~
In Qi~Q experiments were conducted to determine
the bactericidal activity of lysostaphin, mutanoly~in, and
lysozyme compositions toward S. aureus and other mastitis
; pathogen3. The protocol was as follows:
~roSocol fQr Viable Cell A~say~
acterial cell~ (generally 109 cells/ml) from
an overnight plate (incubated at 37C) were resu~pended in
Tris buffer ~20mM Tri~, pH 8).
2. 10 ~1 of bacteria:L cell ~uspension and 1 ml oE
`~ 30 control and teat dip test formulation (i.e. milk, buffer, or ~:
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26704-364/2622
1 ~3075~
buffered detergent etc., containing the lyso~taphin compo-
sition? were combined.
3. The cells were incubated for various times at
37C. `~
4. The bacterial suspensions were centrifuged for
2 minutes in benchtop microcentrifuge to pellet cells.
5. The pellet was washed twice with l.0 ml Phage
.~ . . . ..
buffer. ~ ;
6. The cells were resuspended in 1.0 ml of Phage
buffer, serially diluted in Phage buffer as appropriate, and
,
lO0 ~1 were plated on GL agar (S. aureu~, E. Q~l Kleb3i~11a
pneumoni~e.) or Trypticase Soy agar (S. aqalactiae).
7. The plates were incubated overnlght at 37C
and control and test plates were scored for colony forming
: ~ ,
units, (hereinafter C~U), to determine percen~ survival.
ComDosition of Phaae buffer~
50 mM Tris, pH 7.8; 1 mM MgSO4; 4 mM CaC12; lO0 mM
NaClJ Gelatin, l.0 g/l. (Phage buffer helps stabilize any
protopla3ts and ~pheroplasts that did not lyse durlng
treatment).
-
Com~o~ition of GL aaar Per liter: `~
Difco casàmino acids, 3.0 g; Difco yeast extraict,
3.0 g; NaCl, 5.9 g; Na lactate ~60~ w/v) 9 3.3 ml; 25% ~v/v)
glycerol, 4.0 ml; agar, 15 g; pH adju~ted to 7.8.
Com~Qsition of Try~tica~e Soy aqar per liter:
; ~acto Tryptone, 15 g; ~acto Soytone, 5 g; NaCl,
~ 5 g; agar, 15 9; p~ adjusted to pH 7.3. ~ `~
:~ ~"
Th~ results of in vitro experiments demonstratin~
the bactericidal efficacy of various lysostaphin therapeutic ;~
formulations ace pre~ented in Tables IA-IC. The results are
' ~" " ~',
1 3 3 o 7 5 ~ 26704-364/2622
presented as the percent survivals for S. aureus strains
Newbould 305, strain RN451, the penicillin resistant strains
RN1753 (Penicillinase producer) and Col strain (methicillin
resistant).
Table IA presents results for formulations con-
taining 1 ~g/ml, 0.1 ~g/ml, 0.01 ~g/ml and 0.00 ~g/ml (CNTRL)
lysostaphin. As can be seen from these results all levels of
lysostaphin tested were eective to kill the organisms in
a buffer vehicle (50 mM Tris, pH 8.0). In a milk vehicle,
only 1 ~g/ml and 0.1 ~g/ml reduced bacterial survival.
Table I~ shows the effect of adding a mild non-
ionic surfactant, octylphenoxyl polyethoxy ~10) ethanol,
(Triton X-100), to the lysostaphin Eormulation. For example,
less than 0.001~ of the cells survive exposure to 0.1 ~g/ml
lysostaphin and 0.1~ Triton X-100, while 2.2~ and 7.7~,
respectively, survived exposure to each compound alone. Even
more surprising, less than 0.001~ survival was observed for
0.01 ~g/ml lysostaphin and 0.1~ Triton X-100.
Table IC demonstrates the synergi~tic effect of
ly~o~taphin/penicillin combinations on three strains of
staphylococci. Depending on the doses of each, the combina~
tions of lysostaphin plus penicillin can be 100 to 1000 times
~` more effective than either lysostaphin or penicillin alone
with all threé strains.
Table ID demonstrates the effect of the combina-
;~J
tion of lysostaphin and penicillin compared with their
sequential effect on S. aureu~. $. aureu were suspended at
q
~ 10' cells/ml in milk and incubated for the times indicated in
`~ the table with either lysostaphin and penicillin together or
sequentially. After incubation, samples were centrifuged to
* Trade-mark -12- i ;
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26704-36~/2622
1 33075~
obtain cell pellets which were washed twice, resuspended in
1.0 ml Phage buffer, diluted and 100 ~1 plated on ~L agar.
Colony forming units (CFU) were scored after incubation
overnight at 37C to determine percent survival relative to
S appropriate controls. The lysostaphin/penicillin combina-
tion~ exhibits a synergistically enhanced bactericidal
activity against S. aureus which is at least 3 orders of
magnitude greater than that seen when the two agent~ are
added sequentially.
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~ 26704-364/2622
1 33075~ ~
TABLE IA
The Effect of Lysostaphin On The ~''
Viability of S. Aureus
Incubation ,
S Strain Vehicle Time ~ ~urvival
l.OL 0.lL 0.01L CNTRL ,, ,
S. aureu~ ,--"- -
Newbould 305 Milk 15' 2.8 75.0 100 100 '-~
2h 0.1 82.0100 100 ~' ~
10 RN451 Milk 15' <0.1 22 100 100 - ~-
2h <0.01 41 100 100
Buffer 2h nd 2.2 20 100 ~ ~
TABLE IB ,
. ~ -, .
The Ef~ect Of Non-Ionic Detergent On The Bactericidal
lS Activity of Lysostaphin ~oward S. aureus ~-
Incuba- -
tion
Vehicle Time ~_~Y~YlY9L '~
0.1L 0.01L 0.1~T 0.1L OoOlL CNTRL ,',',~
+0.1~T+0.1~T
S. aureus ~u~fer , ' ~
RN451 +0~13 i` ,; '~`
Triton 2h 2.2 20 7.7 <0.001 ~0.001 100 ,"~
. ~, . ., ~-
TAEILE IC i.`
25 The Effect Of Penicillin On The Bactericidal
,,Ac,t,ivi,ty of Ly~03taphin Toward_S. aureus '~
'i ' 'I' rncul~a' ~
tion '','~,
Strain Vehicle, Time ~_~Y~YiYLl ""` ''' ''
0.1L0.01L 0.1P 0.1L 0.01L CNTRL '~
+0.lP +0.lP ,,'~
S. aureus Milk 30' 19 100 76 2.8 45 100
RN451 2h 26 100 17 <0.01 0.4 100 ,``~i
~lOP) (lOP) (lOP) ' ';
35 RN1753 Milk 2h 1~9 66 46 <0.01 14 100 , ~
penicillinase ,, ~-
positive
(10P) (iOP) (10P)
Col Milk' 2h 1.0 100 67 <0.01 0.5
40 methicillin
resistant ~ ~ -
. ~ :
~` :
~670~--64/~6~
- 1 33075~
TABLE ID
A Co~parison of the Effect of the Combination of
Lysostaphin and Penicillin Versus Their Sequential
Effects on the Survival of Staphylcoccu~ aureus
5(Strain RN451) in milk at 37C
combo(2h) lspn(2h) pen(2h1 Pen(2h~/ lspn(2h)/ ~:
lspn(0.5h) pen(O.Sh)
~survival 0.0005 23 25 0.3 10
l~pn = lysostaphin; pen = penicillin
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26704-364/2622
1 33075~ `
In addition, assays for ly~ostaphin, mutanolysin,
and lysozyme activities which measure the decrease in
turbidity at 600 nm of suspensions of live S. aureus,
S. aqalactiae, and E. coli or Klebsiella_Pneumoniae, respec-
tively, indicated that chelating agents (e.g., EDTA) poten-
; tiate the lytic activity of each of the three bacteriolytic enzymes.
The data indicate that lysostaphin is a rapidly
acting, highly effective staphylocide, the bactericidal
activity of which ls potentiated more than 1000 times by
penicillin or the mild surfactant, Triton X-100. The inclu- ``
sion of a chelating agent further potentiates the bacteri-
cidal activity of lysostaphin. It is also believed that
synthetic p~nicillins and cell wall-active antibiotics will
lS potentiate the activity of lyso~taphin. Lysostaphin is an
eEfective staphylocide in milk, but in buffer the bacteri-
cidal activity of lysostaphin is approximately 10 time~ that `
observed in mllk. ` ~
Example S ~ `
According to ~he general pro~ocol described in
Example~ 1-4, further in vitro experiments were performed to
evaluate the bactericidal activity of a lyso~taphin compo~
tion comprising bacteriolytic enzymes, a non-ionic detergent
and buffered chelating agent. As shown in Table II a
25 formulation containing 1% Triton X-100, 0.1 ~g/ml lysos~
taphin, 10 ~g/ml ly~ozyme, and 5 mM EDTA in 20 mM Tris, pH
; 8.0, (AMBI Teat Dip-0.1) was ex~remely effective against a
wide range of mastiti3-causing pathogens, including S aureus
strain Newbould 305, S. epi~ermidi~, Streptococcu9 aqalactiae
~ ~ ' '' ;
-16
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1 33075
. :~
strain McDonald and strain C48, and clinical isolates of
Streptococcus uberis, E. coli, and Klebsiella ~neumoniae.
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~ 26704-364/2622
1 33075~
TABLE II
In Vitro Efficacy Of AMBI Teat Dip-l
. Aqainst Mastitis Pathoqen3
Strain Viable Count ~ Survival ~ .
: : '
5 Staphylococcus aureus 7.0 x 105 <0.001
. (Newbould 305) ~ -
Staphylococcus aureus 5.7 x 105 <0.001
~N451) :~
Staphylococcu~ epidermidi~ 8.3 x 105 . <0.001
10 (PS) , ::: :
. Streptococcus agalactiae 3.9 x 105 <0 001
(McDonald)
,~
Streptococcus agalactiae 2.9 x 104 <0.001
(C48)
15 Streptococcus uberis 6.9 x 105 <0.001
l (PS), ' ~
Escherichia coli 9.1 x 105 <1.0
Kleb~iella pneumoniae 9.6 x 105 <1.0 `.
20 tPS) ; ~`~
"~
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" .
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Example 6
Trials on cows were performed which demonstrated
the efficacy of lysostaphin teat-dip compositions in vivo.
The tests were performed generally according to Protocol A
of the National Mastitis Council. In general, teats were
cleaned with a 1% iodine wash solution and dried with a
paper towel. Teats were then rinsed with alcohol and allowed
to air dry. All four teat~ per cow were next dipped in a
cell/ml suspension of S. aureus strain Newbould 305 to
CGver 1/2 the teat, and allowed to air dry for 30 minutes.
Two teats (right fore and left rear~ were then dipped in a
lysostaphin test teat dip formulation (10 ~g/ml lysostaphin
in 0.85~ saline) to cover 2/3 of the teat, and allowed to air
dry or 30 minutes; the remaining two teats acted a non~
treated controls. Each teat was ~irst swabbed with a moist
cotton ~wab and then washed with 10 ml of 0.85% sterile
saline solution; the wash was collected into a sterile 30 ml
tube. A 0.2 ml sample of the wash, and appropriate dilutions
thereof, were plated on blood a~ar in duplicate and incubated
at 37C for 24-48 hours, Colony forming unit~ were deter-
mined and percent survival of S. aureus calculated relative ~;
; . " I ~ i
to controls.
Ten ~g/ml solutions of lysostaphin in 0.85% salinecompletely di~inected invading S. aureu~ ~rom cow teat
surfaces. Moreover, lyso~taphin applied to teat surfaces
prior to exposure of teats to $. aureu~ ~u~pension~ had
,,. . ~ .
sufficient residual activity on the teat ~urface to prevent
-':- :.
colo~ization o~ the teat. Residual activity could be ;~
enhanced by inclu~ion of a polymeric adsorbent and/or inert
30 carrier protein to reduce lyso~taphin wash-off. `
:"'`:'. ;'~. ~
--19 ~
~ ~ .
~67~-364/26Z2
1 33075~
Example 7
In accordance with the results from Example 6 and
the data obtained in vitro, an enhanced teat dip formu-
lation (AMBI Teat Dip 1.0) comprising 1.0 ~g/ml lysostaphin,
lO ~g/ml lysozyme, 1.0 % Triton X-100, and 5 mM EDTA in
20 mM Tris buffer, pH 8.0 was evaluated as a disinfectant
against S. aureus ~train Newbould 305. Teats were dipped in
lO~ cells/ml S. aureys strain Newbould 305, and allowed to ;~
` air dry for 30 min. The treated teats were then dipped in
AMBI test teat dip-l.0 solution (1.0 ug/ml lysostaphin,
10.0 ~g/ml lysozyme, l.0~ Triton X-100, 5 mM EDTA, 20 mM Tris
buffer, pH 8.0j and allowed to air dry Eor 30 min. Teats
were swabbed with a moi~t cotton swab, and rinsed with 10 ml
sterile 0.85% saline~ The swab and rinse were plated sepa-
rately on blood agar plates, incubated 24-48h and CFU deter-
mined. The results, shown in Table IIIA clearly demonstrate ;~
..
the eEficacy oF this preparation. At least a 3 fold order of
magnitude reduction was observed in the numbers of S. aureu5
¦ recovered from treated teats; 50~ of treated teats were free ~ -
~rom invading S. aureus.
Corresponding tests were performed in which teats
were dipped in preparatiohs containing 2 x 107 cells/ml ! '
StreDtococcus ~galactiae strain McDonald, and then allowed to
air dry ~or thirty minutes. The results of ~hese tests are
25 shown in Table IIIB. All of the treated teats were free of ~ `~
`~ S. a~alactiae.
'': '.'` ~'
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~ 26704-364/2622
~ ~ 1 3 ~ 0 7 5 ~
TA~LE IIIA
In Vivo Efficacy of ~M~I Teat Dip-1.0 Against
, Staphylococcu~ aureui~ On Cow Teats ~ :
CONTROL CONTROL
5 C~U's ~er ml CFU~s per ml 1
Cow No. LF ~ RH LH ¦ RF
: ~ '
1 225 1,675 13 o
2 24r500 19,500 8 175
10 3 300 15,000 0 15 `'~
4 78 155 0 150 -: :
. 5 50,500 18,750 5 8 : ,
6 44,Z50 65,500 0 0
7 75 43 35 3 ;~
lS 8 175 1,150 o 0
9 68 5~ 0 5
550 300 0 0
Average 12,072 12,213 6 36
Total Qtrs
20Negative 0/10 0/10 6/10 4/10
',~'~ ;~``,
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~ -21~
~ 26704-364/2622
~ 133075~
TABLE IIIB
In Vivo Eficacy of Teat Dip-1.0 against Streptoeoccus
a~alactiae (McDonald strain) on Cow Teats
Cow. No. CONTROL TREATED
CFU's per ml CFU's per ml
LF RH LH RF
- 1. 5 lS 0 0 .-
2. 53 360 0 0
3. 115 48 0 0
4. 150 10 0 0
5. .13,750 1,200 0 0
6. 16,250 725 0 0
7. 95 320 0 -~
8. 0 450 0 0
9. 1,175 775 0 0
10. 150 300 0 0 ;;::~
. - - - `:
Average 2,574 420 0 0
Total ~trs
20 negatlve: 1/10 0/10 10/10 10/10
: ~ - ~-~- -- - -- - . _ -;
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~:
.
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26704-364/2622
,~
1 33075~ `
Example 8
Guinea pig mammary glands were infected with 200-
300 CFU of S. aureus strain Newbould 305. Thre~ days post-
infection, the gland~ were infused with a single dose of :
ly~ostaphin dis~olved in 200 ~l 0.85~ sterile saline. Milk
samples were collected from the glands 6 hours aftec treat-
ment and at 12 hour intervals thereafter for at least 5 days
after treatment. 100 ~1 milk sample~ from treated and -~
nontreated infected glands were plated on blood agar. After
lO 24-48 hour~ incubation, the plate~ were counted to determine ~:
CFU. The single do~es of lysostaphin which were sufficient
to eliminate the infection did not produce adverse side
efects and indicated that intramammary infusions of lysos- - -
taphin are effectlve again~t staphylococcal mastitis. At 125 ~ ~
15 ~g/kg, glands were cleared of infection by the 6 hour post- ~`
treatment sample and remained clear throughout the study.
," . , .:
_~ ,
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, -23- ~
:,: .
TABLE IV
Efficacy of Intramammary Infusion of Lyostaphin Toward
Experimental STAPHYLOCOCCAL Mastitis in Guinea Pig
<IMG>
-24-
1 3307 5~
It can be seen from these examples that
lysostaphin is effective for treatment of staphylococcal
mastitis and that its effect is greatly enhanced when used
in combination with penicillin or with substances such as
mild surfactants and chelating agents.
Production of Lysostaphin from Bacillus
Lysostaphin for use according to the claimed
invention can be obtained from either natural or
recombinant sources. Preferably, the lysostaphin is
lo obtained from cultures derived from Bacillus sphaericus
strain oo transformed by recombinant plasmids which direct
lysostaphin synthesis as described in U.S. Patent
4,931,390. This method provides for both high levels of
lysostaphin production substantially free from staphyloccal
immunogenic contaminants. Lysostaphin purification is
facilitated since active lysostaphin accumulates directly
in the growth medium. Using this method, Bacillus
sphaericus Oo transformants containing plasmid pBC16-lL (B.
s~haericus OO/pBC16 lL) have been found to be particularly
suited for the purpose, although other transformed Bacillus
strains are also useful as a source of lysostaphin. One
method for obtaining lysostaphin from micro-organisms
transformed by recombinant plasmids conkaining the gene
which codes for lysostaphin is fully disclosed in U.S.
Patent 4,931,390 (Recsei).
The lysostaphin-producing organism is grown under
conditions conducive to the production of lysostaphin. The
optimum conditions will vary from strain to strain;
however, certaih types of growth media and fermentation
conditions are known to enhance lysostaphin production. In
the case of the Bacillus sphaericus OO/pBC16-lL
transformant, the preferred growth medium is VY broth (25g
Veal Infusion ~ 5g
. . :
}~
26704-364/2622
1 33075'~
Yea~t Extract/liter ) under well-aerated condition~ ( ~ee
Table V).
: '
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- 2 6~
26704-364/2622
. .
1 33075~3
TABLE V
Effect of Aeration on Lysostaphin Production
b~ the Bacillus SPhaericus OO~PBC 16-lL Transformant
Stirrinq SPeed
5Klett 100 rpm _ (Fluted2 320 rpm
250 21.8 36.2 35.9 30.0
350 4~.1 68.9 45.3 ~5.0
400 88.5 62.7 102.~ 71.4 ~`
~ 10 450 n/a 86.4 52.3 135.9
3 O/N 64.4 31.3 37.6 57.5
: ~ '` .; .
Cultures (40 ml) in 300 ml Klett flasks were inoculated with ~ ~
4 ml of overnight primary culture. Growth medium: VY broth ` ~ '
containing 5 ~g/ml erythromycin.
Sample3 were removed at times throughout growth. Super~
natant3 were assayed or lysostaphin activity by turbi-
dometric clearing o~ dead cell su~pensions of S. aureus.
Results are presented as ~9 lysostaphin per ml.
. " ~
. , ,
: :; ~
. ~ . .
26704-364/2622
.
1 33075~3
~. s~ha~xicus 00/pBC16-lL transormant grown on VY medium
produced and secreted approximately 130 mg lysostaphin per
liter of culture medium, which is more than four times the
amount produced by S. simulans under the best fermentation
conditions currently available. Lysostaphin accumulates in
the growth medium with little or no degradation, even after
prolonged incubation of cultures, and accounts for more than
80~ of total extracellular protein.
Lycostaphin i3 i~olated from the growth medium in
accordance with known fractional precipitation (salting out)
procedures. Alternatively, a particularly effective puri-
fication is achieved by combining a precipitation and a
chromatographic separation of the fermentation broth from
culture~ of the lysostaphin-producing _. s~haericus
lS 00/p~C16-lL transformant.
Cells are removed from the fermentation broth, for ~ ;
example by centrifugation or ultrafiltration, and solid
ammonium sulfate is added to the supernatant to 40-60
preferably 50%, o~ ~aturation. After 1 hour at 4C, the
ly~ostaphln-contalning precipitate i9 recovered by centri-
fugation. Recovery at this step is greater than 80~.
~ he precipitateii~ redissolved in a minimum volume
of 10 mM sodium phosphate buffer (pH 7.00, S0 mM NaCl) and
dialyzed again~t 100 volumes of the ~ame buffer. After
removal of any particulate material, the dialyzed ~olution is
chromatographed on a cation exchange column (preferably `
Pharmacia FPLC Mono S) and eluted using a buffered gradlent -~
of in~reasing salt concentration from O.OS to 0.25 M NaCl.
Recovery of lysostaphin for the 3ingle chromatographic step
30 was more than 90%~ Lysostaphin activity i5 associated with ~`
,~
-28-
~- '; :'
26704-364/2622
1 330158
two major peaks (Fig. 1). The later eluting peak of lysos-
taphin is comprised of non-covalent aggregates of the
protein. These aggregates dissociate on dilution in buffer
and under conditions of sodium dodecylsulfate polyacrylamide
gel electrophore~is.
Construction of the plasmid vector pBC16-lL
which contains the ~ne codinq for lysostaphin
Ly~ostaphin-producing strains of Bacillus sphaericus
,~
can be produced using recombinant DNA techniques and prefer-
ably those described in copending applications 852,407 and
034,464. Specifically, total S. simulans DNA is partially ~-
cleaved by the appropriat~ restriction endonuclease and DNA
fragments 50 generated are then ligated to a linearized known i-
vector tpUC8) with compatible ends, carrying an antibiotic
resistance marker and the lac Z' gene (i.e. B-galactosidase
gene). The ligation mix is then transferred to E. coli
~JM105) by transformation. Successful insertions of the
lysostaphin gene into the plasmid can be found by selecting
fo~ transformants by growth on the appropriate antibiotic,
and then ~inding those with a la~ Z' negative phenotype.
Lysostaphin production is detected by turbidometric clearing
of a suspen~ion o~ ~L_~Y~ either in solution ~ormat or as
an overlay on agar plates. ~ ;
Using various lysostaphin-producing E. coli JM105
transformants, restriction analysis and subcloning of the
JM105 plasmid DNA showed that the DNA ~equence coding for
ly30~taphin was localized to a 1.5 kbp Hpa II~Hind III DNA
fragment. This fragment was vi~ualized after electrophoresis
by ethidium bromide staining and transferred to a nitro~
30 cellulose filter strip. The strip was washed with NET buffer ~;
~0.15 M NaCl, 0.1 m EDTA, 0.02 M Tris, pH 8.0) and the ;
-29- ;
26704-364/2622
1 3~0~5~3
tran~ferred DNA was eluted by incubation of the strip in NET
buffer ~ontaining 1 M NaCl for 1 hour at 65C, Ethidium bro-
mide was removed from the DNA by extraction with n-butanol.
DNA, precipitated by addition of two volume~ of cold 95~
ethanol to the aqueous phase, was collected by centrifuga-
tion, washed with 80% ethanol and dissolved in TE buffer
(10 mM Tris, 1 mM EDTA, pH 8.0~. Recombinant plasmid~ capa-
ble of transforming . subtilis as well as B. sphaericus to
expres~ lysostaphin were constructed using a derivative
of plasmid pBC16 (pBC16-1) as a cloning vector. p~C16 is a
tetracycline resistant (Tetr) Bacillus plasmid, oriqinally
isolated from cereus (K. ~ernhardr H~ Schremph, and
W. Goebel, 133 J Bac~. 897, 1978). Plasmids indi tinguish- -
able from p~C16 by re~triction analy~is and Southern Hybridi-
5 zation were also found in soil isolates of ~. subtil g and
b~L~i~Ya (J. Polak and R.N. Novick, 7 Plasmid 152,
1982). ~ ~ ~
The p~C16 derivatlve (p~C16-1) used a~ the cloning ~ ~-
vector wa~ con~tru~ted by ligating the TaqIA Fragment of ~;
plasmid pE194 (~. Neisblum, M.Y. Graham, T. Gryczan, and
D. Dubnau, 137 ~. ~a$t. 635, 1979), an erythromycin resistant
(ermr) plasmid from ~ , with a partial TaqI digest of
plasmid pBC16 using T4 Ligase. AEter tran~fer of the liga~
tion mixture to . s~tilis by protopla~t tran~formation
(S. Chang and S.N. Cohen, 168 Molçc._Gen Genet. 111, 1979),
clone~ that were reslstant to both tetracycline and erythro-
mycin were selected. One such clone was designated pBC16-1.
~ estriction analy~is revealed that pBC16-1 con-
taine~ all of the pBC16 TaqI fragments plus the TaqIA frag-
ment of pE194 whieh contains the erythromycin resistance
',.
-30- ~
26704-364/2622
` :
1 33075~
determinant. p~C16-1 was then digested with the restriction
endonuclease PvuII, thereby removing about 25% of the plasmid
DNA including mo~t of the tetracycline re~istance determi-
nant. The Pvu II-digested vector pBC16-1 was treated with
calf intestinal alkaline pho~phatase. The 1.5 Kbp DNA
fragment which codes for lysostaphin was treated with the
Klenow fragment of DNA polymerase. The 1.5 Kbp DNA fragment
and plasmid DNA were then mixed and ligated using T4 ligase,
and the ligation mixture was tran~ferred to B. subtilis by
protoplast transformation. Transformants were resistant to
erythromycin, sensitive to tetracycline, and produced ly~o-
staphin as indicated by zones of clearing when grown on agar
containing dead S. aureus cells. One such lysostaphin pro-
ducing clone was picked and desi~nated 3L_gg~i~L~/pBC16-lL.
Plasmid pBC16-lL DNA extracted from the B. subtilis/pBC16-lL
tran~formant was isolated after ultracentri~ugation in an
ethidium-bromide cesium chloride density gradient. Plasmid
pPC16-lL DNA was tran~erred by protoplast tran~formation to
various species o~ ~l~_us, including B. s~ericus strain
20 00. Tranqformants were re~istant to erythromycin and pro- `~
duced lysostaphin. The B. sphaericus OO/pBC16-lL trans-
formant provides maximum production of lysostaphin and !
permit accumulation of intact, enzymically active product.
~. sph~ericu~ strain 00 was originally isolated from soil
and is maintained in the culture collection ~RN3106) of
the Public Health Research In-~titute, New York, New York.
B. s~ cus OO/pBC16-lL i9 maintained in the culture
collection of the Public Health Research Institute, New York,
New York and has been depo~ited with the American Type ~ ;
Culture Collection under ATCC Accession No. 67398.
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