RESISTANCE PATTERNS, PLASMID PROFILES AND pLACTAMASES OF CLINICAL ISOLATES OF
PSEUDOMONAS AERUGINOSA
BY
M.F. Ghalya, E.Y. Tohamy", M.S. Abdel-SabourC , A.A. Kadryh ,
M. Abou El-Hawa" and A.M.A. Zaid"
FROM
a
Dept. of Botany, Faculty of Science. bDept. of Microbiology, Faculty of
£ Pharmacy , Zagazig University and Dept. of Genetics, Faculty of Agriculture
Moshtohor , Benha branch , Zagazig University , Egypt .
ABSTRACT
A total of 94 clinical strains as well as two standard strains of Pseudomonas
aeruginosa were tested for their susceptibility against 11/3-lactam antibiotics. All
isolates were resistant to at least 6 antibiotics including: ampicillin, amoxycillin,
amoxycillin/clavulanic acid, cephalexin, cephaloridine and cioxacillin. Ceftazidime ,
aztreonam and/or imipenem, exhibited the highest activity with resistance rates of
2.1% and 3.1% respectively, followed by cefotaxime (14.6%) and ceftriaxone
(38.5%). Isolates no. 88 and 90 were resistant to all antibiotics under investigation
except imipenem. The resistant patterns of P. aeruginosa isolates were heterogenous,
since as many as 8 different patterns were recognized among the tested isolates, filactamases from twelve mostly resistant isolates of P. aeruginosa were analyzed by
isoelectric focusing(IEF). Six isolates from the twelve multiresistant P. aeruginosa
isolates revealed the same pattern of /3-lactamases with isoelectric focusing point
ranging from 7.5-8. 7. However, the highest production of (3-lactamases were
detected in isolates no. 33, 90 and'94, these isolates revealed different resistant
patterns. The same multiresistant isolates used in isoelectric focusing were screened
for their plasmid profiles. The results indicated that P. aeruginosa isolates with the
same plasmidprofiles showed different resistant patterns.
INTRODUCTION
Pseudomonas aeruginosa is
one of the major opportunistic pathogens. These bacteria cause several infections which
are associated with urinary, respiratory tract, burns, wounds, eyes as well as ear infections
(Ashour. 1980, Kadry, 1985, Boukadida etal., 1991,
Levine & Niederman, 1991, El-Dally, 1992 and Ravaoarinoro el al., 1996). P.
aeruginosa significantly contributes to high morbidity and mortility rates in immunocompromised patients including those with cystic fibrosis, neutropena, extended burns
and
Egypt J. Biotechnol. Vol. 10, July, 2001.
291
AIDS (Olson et al., 1984, and Grim wood, 1992).
P. aeruginosa remains the most prominant Gram negative rods causing hospital
acquired infections and a major source of morbidity and mortility in intensive care
units (Yee et al., 1996). The emergence of resistance to the antibiotics specifically
aminoglycoside and P-lactam antibiotics is a major problem in treating infections with
P. aeruginosa (Sanders et al., 1984, Chow et al., 1989 and Radberg et al., 1990).
Resistance to antimicrobial agents may be mediated by genes that are encoded on the
host cell chromosome, plasmids or transposons, which are capable of integrating into
the plasmids and/or the chromosomes (Williams et al., 1988).
The epidemiological studies of P. aeruginosa infections carried out by several
methods including biotypes, serotypes, pyocin production, phage typing and
resistant, patterns (Pvtt, l%8y
Advancing in molecular techniques make use of palsmid DNA profile as an
epidemiologic tool of study (Plesiateffl/., 1988).
Bacterial resistance to P-lactam antibiotics is often associated with the production of
p-lactamases which hydrolyze the cyclic amide bond in the p-lactam ring of
penicillins, cephalosporins and related compounds (Abraham & Chain, 1940, Bush et
al., 1991 and Sotto et al., 1996). P-lactamases are mostly encoded by R-plasmids
(Barthelemy et al., 1992). Most of
these plasmid-mediated P-lactamases are belong to class A (1) and they have been
grouped on the basis of their substrate profiles, isoelectric points and DNA
hybridization (Huovlenen, 1988).
The aim of the present study was to describe the susceptibility of the clinical isolates
of P. aeruginosa to different P-lactam antibiotics, with special emphasis on isoelectric
focusing of p-lactamases and plasmid profiles.
MA TERIALS AND METHODS
Bacterial strains:
Ninety four (94) clinical isolates of Pseudomonas aeruginosa were isolated from
documented infections at Zagazig University Hospitals, Egypt, in February 1997.Out
of these, 25 were isolated from urinary tract infection, 33 from wounds 31 from
respiratory infection, 4 isolates from blood and 1 isolate from eye. All clinical
specimens were transferred to the laboratory, streaked on cetrimide agar (Oxoid)
and incubated at 42 C. Single colonies were streaked separately on nutrient agar
slants, incubated at 37 °C for 24 h, then transferred to a refrigerator until used in
identification. Two control strains (ATCC 27853 and ATCC 9027) from American
Type Culture Collection were involved in this study.
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Egypt J. Biotechnol. Vol. 10, July, 2001.
Identification of Pseudomonas aerusinosa isolates:
The colonies grown on cetrimide agar were identified as P.aeruginosa by traditional
biochemical tests according to Degtyar et a/., 1985, Gilardi, 1991. The P.aeruginosa
isolates were confirmed for identification by using the API 20E analytical profile
index (API-Bio Merieux La Balme Les Grotes, France).
Antibiotic susceptibility test:
The susceptibility patterns of identified P.aeruginosa isolates to different antibiotics
have been carried out according to Bauer-Kirby single disk diffusion method (Bauer
el al.. 1966) on Mueller-Hinton agar plates (Merck, Germany). Antibiotic disks used
were : Ampicillin (AMP) 10 u.g, Amoxycillin (AML) 20 ^ig, Amoxycillin/clavulanic
acid (AMC) 20/10 ng, Cephalexin (CL) 30 u^, Cephaloridine (CR) 30 j^g. Cloxacillin
(OB) 5 ug, Ceftriaxone (CRO) 30 ug, Ceftazidime(CAZ) 30u,g. Aztreonam(ATM) 30
ug. Cefotaxime (CTX) 30 (.tg and Imipenem (IPM) 10 u.g, all these disks were
purchased from Oxoid Co.. England. The diameters of inhibition zones were recorded
and interpreted according to Acar & Goldstein 1991 and National Committee of
Clinical Laboratory Standards NCCLS( 1999).
Detection of 3-Lactamase:
Sonicated extracts of all isolates were prepared from bacterial cells
after culture in brain heart infusion (Oxoid) for 24 h in a shaking incubator at 37 °C,
and p-lactamase detection was carried out by the nitrocefm (Oxoid) assay according
to O'Callaghan el al.. (1972).
Isoelectric focusing:
p-lactamases from twelve multiresistant isolates of P.aeruginosa were focused in
7.5% polyacrylamide (Sigma) gel containing pH 3.5 to 10.0 ampholytes (pharmacia
LKB Biotechnology. Uppsala Sweden) by the method of Minami et al. (1996). The
gel was focused across the width at 4 °C and 1600 volt for 90 min with a mulliphore
2197 power unit (LKB). The pH was measured across the focused gel at 0.5 cm
interval distances. Then the gel was overlaid with a filter paper soaked in 50 ug/ml of
nitocefin solution for 20 seconds.
Plasmid analysis:
Plasmids screening was performed on the twelve multi resistant P. aeruginosa isolates
by the alkaline method (Birnboim & Doly, 1979) which modified by Millesimo et al..
1996. Isolates were grown overnight in 5 ml of luria broth (tryptone : 10.0 g, yeast
extract: 5.0 g, sodium chloride : lOg in 1000 ml distilled water) at 35 °C with shaking.
1.5 ml aliquot was pelletted by centrifugation at 12,000 xg for 10 min. at 4 °C in a
microfuge and resuspended in 100 ul of lysis buffer (50 mM glucose, 10m M EDTA
[pH:8], and 5m M Tris-HCl
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Egypt J. BiotechnoL Vol. 10, July, 2001.
[pH:8] and then incubated for 5 min in ice. Denaturating buffer (0.2 N NaoH, 1%
sodium dodecylsulphate) in a volume of 200 ul was added, the solution was mixed by
inversion and left at 0°C for 5 min. Then 150 ul of 3M potassium acetate were added
and mixed by inverting, then incubated for 5 min and centrifuged at 12,OOOX g for 5
min. The supernatant was transferred to a fresh tube and after phenol-chloroform (1:1
volume) extraction, the DNA was precipitated by adding 1 volume of isopropanol and
spinning at 15000 X g for 10 min at room temperature. The pellet obtained was
washed with 70% ethanol , dried and resuspended in 50 ul lOmM Tris-HCL (pH 7.5).
Agarose Gel Electrophoresis:
Agarose gel electrophoresis technique was employed for the detection of plasmid
DNA present in the tested isolates (12 isolates) of P. aeruginosa according to
Millesima et o/., 1996. The plasmids DNA carried on E. coli /I Hind lU digest were
used as molecular weight markers. The approximate molecular weights of the
unknown plasmids were determined in comparison to the known molecular weight
markers.
RESULTS
Antibiotic susceptibility test:
The susceptibility of Pseudomonas aeruginosa isolates against 11 (5-lactam
antibiotics were
summarized in Table 1. The data revealed that 2 (2.1%) of the isolates were resistant
to ceftazidime (CAZ), 3(3.1%) were resistant to imipenem (IPM), 3(3.1%) were
resistant to aztreonam (ATM), 37 (38.5%) were resistant to ceftriaxone (CRO) and 14
isolates (14.6%) were resistant to cefotaxime (CTX).
All the isolates were resistant to the following antibiotics ampicillin (AMP),
amoxycillin (AML), amoxy-cillin/clavulanic acid (AMC), cephalexin (CL),
cephaloridine (CR) cloxacillin (OB), as shown in Table 1.
The resistance patterns of P. aeruginosa isolates (Table 2), showed that all the
isolates under investigation were resistant to at least 6 of tested antibiotics. Isolates
no. 88 and 90 were carrying resistant determinants for 10 antibiotics. The resistance
patterns of P. aeruginosa isolates were heterogenous, since as many as 8 different
patterns were recognized among the tested isolates Table 2).
3-Lactamase screening assay:
Sonicated extracts of all P. aeruginosa isolates under investigation gave positive Plactamase with nitrocefm.
Isoelectric focusing patterns:
Twelve multiresistant isolates carry at least 6 resistant determinants were chosen for
detection of P-lactamases (Table 2 and Fig. 1).
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Egypt J. Biotechnol. Vol. 10, July, 2001.
Eight isolates produced P-Lactamases of different pi ranging from 7.5-8.7. Six isolates
from these eight p-lactamases producers showed approximately similar patterns of Plactamases with isoelectric points (pi) ranging from 7.5 to 8.7 (Fig. 1). Isolates 66
produced p-lactamase of pi 8.5. Isolate 41 produced P-lactamases (four bands) of pi
ranging from 7.7-8.5. No p-lactamases were detected in the remaining four isolates.
Plasmid screening:
The same multiresistant isolates used in isoelectric focusing were screened for their
plasmid profiles (Table 2). The results indicated that six isolates harbored two
different plasmids with approximate size of 2.03 and 1.58 Mda and one isolate
harbored a single plasmid with approximate size of 3.76 Mda. Five isolates were
plasmidless (Figs 2 and 3).
DISCUSSION
The role of P. aeruginosa as a nosocomial pathogen in hospitalized patients with
critical illness has markedly increased in the past decade. P. aeruginosa has become
the most prominant cause of nosocomial Gram-negative pneumeniae (fcevin and
Niederman, 1991).
A major problem in the treatment of P. aeruginosa
infections is the resistance of these bacteria to a number of structurally unrelated
antimicrobial agents (Mouten et a/., 1993). Hence the knowledge of resistance pattern
is essential, with special attention to the production of P-lactamases, their typing by
isoelectric focusing, and the plasmid profiles for the most resistant isolates.
In this investigation, 94 clinical isolates of P. aeruginosa were identified from
different infection sites. Identification of the isolates was carried out by the traditional
methods (Degtyar et al., 1985, Gilardi 1992) as well as multitest API2QE system.
All clinical isolates of P. aeruginosa were studied for their susceptibilities against 11
p-lactam antibiotics (Table 1). The results revealed that all tested organisms were
resistant to at least 6 antibiotics. Thus the use of these antibiotics may be of limited
value in treating infections with P. aeruginosa . On the other hand, the study revealed
that the most effective drugs under investigation against P. aeruginosa infections
were the third generation cephalosporins namely ceftazidime (CAZ) followed by
imipenem (IPM) and aztreonam (ATM), respectively. The susceptibility tests revealed
that 2.1% of P. aeruginosa isolates were resistant to ceftazidime, 3.1% were resistant
to imipenem, 3.1% were resistant to aztreonam while Al-Zamel et al. (1996) reported
that295
Egypt J. Biotechnol. Vol. 10, July, 2001.
19%, 9% and 21 % of P. aeruginosa isolates were resistant to ceftazidime,
imipenem and aztreonam, respectively. The fundamental cause for the appearance and
spread of antimicrobial resistance has been increasing antimicrobial use. However,
other factors contribute in both inpatient and outpatient settings (Hellinger, 2000).
The resistance of P. aeruginosa isolates to imipenem could be mediated by
diminished outer membrane permeability due to the decreased production of outer
membrane protein D2 (Ochs, et al., 1999). While the emergence of resistance of P.
aeruginosa isolates to ceftazidime and aztreonam is probably attributed to the
derepression of chromosomal cephalosporinase production (Livermore, 1993).
Isoelectric focusing (IEF) is one of the basic methods for identification and
differentiation of P-lactamases (Matthew et al., 1975). The p-lactamases produced by
twelve multiresitant isolates of P. aeruginosa were analyzed by isoelectric focusing
(Table 2 and Fig. 1). Eight isolates from the chosen twelve multiresistant P.
aeruginosa isolates produced p-lactamases of different pi ranging from 7.5-8.7. Six
from these 8 multiresistant P. aeruginosa isolates showed approximately the same
pattern of P-lactamases with isoelectric point (PI) ranging from 7.5 to 8.7. Although
these isolates
have the same types of p-lactamases (Fig. 1), they showed different resistance
patterns. No p-lactamases were detected in the remaining four isolates. The major
band of P-lactamases in the selected multiresistant 8 isolates was detected at
approximately pi 8.7, this P-lactamase was identified as cephalosporinase (Minami et
al., 1996).
Plasmid DNA analysis demonstrated that there is no consistent relationship between
the plasmid profiles and the resistance patterns of the investigated isolates. P.
aeruginosa isolates with the same plasmid profiles showed different resistant
patterns. These results were in accordance with the results of Walia et al.,(l988) and
Abdel-Ghafar et a/.(1994). Variations between the isolates of presence or absence of
plasmids, the occurrence of resistance mechanisms and different phenotypes, this may
be due to the fact that the plasmid can be gained and lost among bacteria (Morlin et
al., 1994 and Stefani & Agodi, 2000).
In conclusion, the clinical isolates of P. aeruginosa were highly resistant to at least 6
of antibiotics under investigation. In addition ceftazidime, imipenem and/or
aztremonam, were the most effective drug against the P.aeruginosa isolates.
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Egypt J. Eiotechnol Vol. 10, July, 2001.
Table (1): The antibiotic susceptibility of Pseudomonas aeruginosa isolates.
Resistant (%)
Con.
jag/disc
Code
Antibiotic
96(100%)
10
AMP
Ampicillin
96(100%)
20
AML
Amoxycillin
96(100%)
30
AMC
Amoxycillin/clavu-lanic acid (2:1)
96(100%)
30
CL
Cephalexin
96(100%)
30
CR
Cephaloridine
96(100%)
5
OB
Cloxacillin
37 (38.5%)
30
CRO
Ceftriaxone
2(2.1 %)
30
CAZ
Ceftazidime
3 (3.1%)
30
ATM
Aztreonam
14(14.6%)
30
CTX
Cefotaxime
3(3.1%)
10
IPM
Imipenern
Table (2):
Resistant patterns, isoelectric focusing patterns and plasmid
profiles of tested Pseudomonas aeruginosa.
Isolate
Resistance patterns
Isoelectric
focusing Plasmid profiles*
numbers
patterns (pi)
(=M.wt) Mda
1
AMP, AML, AMC, CL, CR, OB 7.5.7,7,7.9,8,8.1,
8.5,8.7
2.03 , 1.58
2
AMP, AML, AMC, CL, CR. OB, 7.7,7.9,8.8.1,8.5, 8.7
CRO, CTX
2.03 , 1.58
31
AMP, AML, AMC, CL. CR, OB, CRO, CTX
2.03, 1.58
33
AMP, AML. AMC, CL, CR, OB, 7.5,7.7,7.9,8,8.1,
CRO, ATM, CTX
8.3,8.5,8.7
-
39
AMP, AML, AMC, CL, CR, OB, CRO, CTX
-
41
AMP, AML, AMC, CL, CR, OB, 7.7,7.9, 8.5, 8.7
CRO, CTX
2.03 , 1.58
66
AMP, AML, AMC, CL, CR, OB, 8.5
CRO, IPM
-
87
AMP, AML, AMC, CL, CR, OB, CRO, CTX, IPM
-
88
AMP, AML, AMC, CL, CR, OB, CRO, CAZ, ATM, CTX
-
90
AMP, AML, AMC, CL, CR, OB, 7.5,7.7,7.9,8,8.1,
CRO, CAZ, ATM, CTX
8.3,8.5.8.7
3.76
94
AMP, AML, AMC, CL, CR,OB, 7.5,7.9,8.1,8.3, 8.5,8.7 2.03, 1.58
CRO, CTX
96
AMP, AML, AMC, CL, CR, OB, 7.5,7.7,7.9,8,8.1,
CRO, CTX
8.5,8.7
2.03, 1.58
Abbreviations: ( ~ ) : Piasmidless or p-lactamase absent.
P =Penicillin G; DP = Methicillin; AMP= Ampicillin; AML = amoxycillin;
AMC = amoxycillin/clavulanic acid (2:1); CL = Cephalothin; CR= Cephaloridine;
OB = Cloxacillin; CRO = Ceftriaxone; CTX = Cefotaxime; ATM = aztreonam;
CAZ = Ceftazidime; IPM = Imipenem.
'
*Indicates the number and molecular weight of plasmids in each isolate.
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Egypt J. BiotechnoL Vol. 10, July, 2001.
REFERENCES
Abdel-Ghafar, A.F.; Ashour, M.S.; Abdel-Latif, H.K.; Kadry, A.A.; Atia, A. and
Fekry, S. (1994): Resistance pattern of clinical isolates of Salmonella and its
correlation with plasmid profiles. Zagazig J. pharm. Sci. 3 :51-57.
Abraham, E.P. and Chain, E.
(1940): An enzyme from bacteria able to destroy penicillin. Nature 146: 837.
Acar, J.F. and Goldstein, F.W. (1991): Disk susceptibility test. In Antibiotics in
Laboratory Medicine. Ed, V. Lorian. Baltimore: Williams and Wilkins, pp 17-52.
Al-Zamel, F.A.; Tawfik, A.F.; Al-Shammary, F.J.; El-Kersh, T.A.; Kambat, A.M.
and Shibl, A.M. (1996):
Antibiotic resistance and serotypes of clinical isolates of Pseudomonas aeruginosa in
Riyadh. Med. Sci. Res. 24: 103-105.
Ashour, M.S. M. (1980): Studies on the therapy of burns in experimental animals
subjected to infection with Pseudomonas aeruginosa isolated from cases in Egyptian
hospitals.Ph.D. Thesis, College of Pharmacy, Cairo University, Egypt.
Barthelemy, M.; Peduzzi, J.; Bernard, H.; Tancrede, C. and Labia, R. (1992):
Close amino acid sequence relationship between the new plasmid-mediated extendedspectrum P-latamase MEN-1 and chromosomally encoded enzymes of Klebsiella
oxytoca. Biochim. Biophys. Acta. 1122: 15-22.
Bauer,
A.W.; Kirby, W.M.M.; Sherris, J.C. and Turck, M. (1966): Antibiotic
susceptibility testing by a standarized single disc method. Am. J. Clin. Pathol. 45:
493-496.
Birnboim, H.C. and Doly, J. (1979):
A rapid alkaline extraction procedure for screening recombinant DNA. Nucleic Acid
Res. 7: 1513-1523.
Boukadida, J.; Montalenbert, M.; Gillard, J.L. and B ere he, P. (1991): Outbreak
of gut colonization by Pseudomonas aeruginosa in immunocomprornised children
undergoing total digestive decontamination: analysis by pulsed-field electrophoresis.
J. Clin. Microbiol. 29: 2068-2071.
Bush, K.; Flamm, R.K.; Ohringer, S.; Singer, S.B.; Summerill, R.B. and Bonner,
D.P. (1991):
Effect of clavulanic acid on activity of (3-lactam antibiotics in Senatia marcescens
isolates producing both a TEM-P-lactamase and a chromosomal cephalosporinase.
Antimicrob. Agents Chemother. 35: 2203-2208.
Chow, A.W.; Wong, J.; Bartlett, K.H.; Shafram, S.D. and Stiver, H.G. (1989):
Cross-resistance of Pseudomonas aeruginosa to cipro-floxacine, extended-spectrum
p-lactams, and aminoglycosides and susceptibility to antibiotic combinations.
Antimicrob. Agents Chemother. 33: 1368-1372.
300
Egypt J. BiotechnoL Vol. 10, July, 2001.
Degtyar, N.V.; Litovchendo, P.P.; Znatnensky, N.A.; Abdu El-Hawa, M. W.;
Nazaarchulk, L.V. and Maximets, A.P. (1985): Identification of Pseudomonas
and similar Gram-negative glucose non-fermenting bacteria using a minimal
number of tests. Lab. Delo. 5: 311.
El-Dally, S.A.E. (1992): A study on the proteolytic activity of pathogenic
Pseudomonas aeruginosa stains. M.Sc. Thesis, Faculty of Pharmacy, Zagazig
University, Egypt.
Gillardi, G.L. (1991): Pseudomonas and related genera. In Balows, A., Hausler, Jr.,
Hermann, K.L., Isenberg, H.D. and Shadomy, H.J. (eds), Manual of clinical
microbiology, 5l ed. Washington,DC: American Society for Microbiology 429-441.
Grimwood,
K.
(1992):
The
pathogenesis of Pseudomonas aeruginosa lung infections, in cystic fibrosis. J.
Paediat. Child. Health, 28:4.
Hellinger, W.C. (2000): Confronting the problem of increasing antibiotic resistace.
South Med. J. 93: 842-848.
Huevinen,
S. (1988): Rapid isoelectric focusing of plasmid-mediated P-latamases
with pharmacia-phast system. Anti microb. Agents Chemother. 32: 1730-1732.
Kadry,
A.A. (1985): Antibiotic inactivating enzymes of certain strains of
Pseudomonas aeruginosa. Faculty of Pharmacy, Zagazig University, Egypt.
Levine, S.A. and Niederman, M.S. (1991): The impact of tracheal incubation on host
defenses and risks for nosocomial pneumonia. Clin. Chest. Med. 12: 523-543.
Livermore, O.M.
(1993):
Determinants of the activity of (3-lactamase inhibitors combinations. J. Antimicrob.
Chemother. 31: 9-21.
Matthew, M.; Harris, A.; Marshall, M.J. and Ross. G.W.
(1975): The use of analytical isoelectric focusing for detection and identification of Plactamases. J. Gen. Microbiol. 88: 169-178.
Millesimo, M.; Intinis, G.D.; Chirillo, M.G.; Mussot, T. and Savoia, D. (1996):
Pseudomonas aeruginosa clinical isolates: serotypes, resistance phenotypes and
plasmid profiles. Eur. J. Epidemiol 12: 123-129.
Minami, S.; Akama, M.; Araki, H.; Watanabe, Y.; Narita, H.; lyobe, S. and
Mitsuhashi, S. (1996): Imipenem and cephem resistant Pseudomonas aeruginosa
carrying plasmids coding for class B p-lactamase. J. Antimicrob. Chemother.
37:433-444.
Morlin, G.L.; Hedges, D.L.; Smith, A.L. and Urns, J.L. (1994):
Accuracy and cost of antibiotic susceptibility testing of mixed morphotypes of
Pseudomonas aeruginosa. J. Clin. Microbiol. 32: 1027 -1030.
Mouton, J.; Hollander, J. and Horrevorts, A. (1993):
Emergence of antibiotic resistance among Pseudomonas aeruginosa isolates from
patients with cystic fibrosis. J. Antimicrob. Chemother. 31: 919-926.
301
Egypt J. Biotechnol. Vol. 10, July, 2001.
National
Committee for Clinical Laboratory Standards. (1999): Performance
standards for antimicrobial susceptibility testing. Document M2-A6 and M7-A4.
January 1999. Pennsylvania, USA.
O'Callaghan,
C.H.; Morris, A.; Kirby, S.M. and Shingler, A.H. (1972): Novel
method for detection of P-Lactamases by using a chromogenic cephalosporin
substrate. Antimicrob. Agents Chemother. 1: 283-288.
Ochs, M.M.; McCusker, M.P.; Bains, M. and Hancock, R.E.W. (1999): Negative
regulation of the Pseudomonas aeruginosa outer membrane porin oprD selective for
imipenem and basic amino acids. Antimicrob. Agents Chemother. 43: 1085-1090.
Olson, B.; Weinstein, R.A. and Nathan (1984):
Epidemiology of endemic Pseudomonas aeruginosa : why infection control efforts
have failed../. Infect Dis. 150: 808-816.
Pitt, T.L. (1988): Epidemiological typing Pseudomonas aeruginosa. Eur. J. Clin.
Microbiol. Infect. Dis. 7: 238-247.
Plesiat, P.; Alkahalaf, B. and Michael-Braind, Y. (1988):
Prevalence and profiles of plasmids in Pseudomonas aeruginosa. Eur. J. Clin.
Microbiol. Infect. Dis. 7: 261-264.
Radberg, G.; Nilsson, L.E. and Svensson, S. (1990):
Development ofquinolone-imipenem cross resistance in Pseudomonas aeruginosa
during exposure to ciprofloxacin. Antimicrob.
Agents Chemother. 2142-2147.
34:
Ravaoarinoro, M.; Mohapatra, S.; Shore, J.; Rawal, S.; Omri, A.; Yaghi, J. and
Oriol, F. (1996): Serotyping clinical isolates of Pseudomonas aeruginosa in relation
to infection site, antibiotic susceptibility and beta-lactamase production. Inter. J. of
Antimicrob. Agents Agents. 7: 65-68.
Sanders, C.C.; Sanders, Jr.W.E.; Goering, RV. and Werner,
V. (1984): Selection of multiple antibiotic resistance by quinolones, beta-lactams and
aminoglycosides with special reference to cross resistance between unrelated drug
classes. Antimicrob. Agents Chemother. 26: 797-801.
Sotto,
A.; Brunchwig, C.; O'Callaghan, D.; Ramuz, M. and Jour dan, J. (1996):
In Vitro evaluation of P-Lactamase inhibition by latamoxef and imipenem. J.
Antimicrob. Chemother. 37: 697-701.
Stefani, S. and Agodi, A. (2 000):
Molecular epidemiology of antibiotic resistance. Internat. J. Antimicrob. Agents,13:
143-153.
Walia, S.; Madhavan, T.; Williams, T. (1988): Protein patterns, serotyping and
plasmid DNA profiles in the epidemiologic figerprinting of Pseudomonas aeruginosa.
Eur. J. Clin. Microbiol. Infect. Dis. 7:284-255.
Williams, H.; King, A.; Shannon, K. and Phillips, I. (1988):
Amoxycillin/clavulanate resistant Escherichia coli. Lancet 1: 304-305.