The relationship between biotype, serotype, antibiotic susceptibility and coa gene polymorphism in Staphylococcus aureus isolated from bovine

Abstract

Abstract: Forty Staphylococcus aureus strains of different biotypes and serotypes, previously isolated from bovine, were used in this study. The presence of coagulase gen (coa) were investigated using PCR assay. coa gene shows polymorphism since it appear in 500, 600, 650, 800 and 850 bp. The relation between genotype, biotypes and serotypes of S. aureus were also investigated. The relation between biotype and serotype of S. aureus isolates revealed that the high occurrence of serotype IV was recorded in the biotype C. No serotype I or II were recorded in biotype A isolates. Serotype V not recorded in any strain. The relation between the biotype, genotype and serotype in the same forty S. aureus isolates, showed that the isolates from biotypes C and A, revealed that the same product of coa gene (500 and 800 bp) share in the same serotype, Π and IV. The study of the relation between the coa product with oxacillin showed that the total resistance in genotypes to oxacillin was 50%. The high percentage of resistance was found in isolates with coa product 850 bp followed by 500 bp. The relations appeared between the coa product with vancomycine resistance in 40 bovine isolates were also recoded. The total resistance in genotypes to vancomycine was 50%. The high percentage of resistances was found in isolates with coa gene product 850 bp.
Key words: S. aureus, coa gene, VRSA, ORSA

Full text

17 Sci. Cong. 2016, Fac. Vet. Med., Assiut Univ., Egypt
33
College of Veterinary Medicine, University of Basrah, Iraq; College of veterinary
Medicine, University of Wasit, Iraq
THE RELATIONSHIP BETWEEN BIOTYPE,
SEROTYPE, ANTIBIOTIC SUSCEPTIBILITY AND
COA GENE POLYMORPHISM IN STAPHYLOCOCCUS
AUREUS ISOLATED FROM BOVINE
BY
BASIL A. ABBAS, MOHAMMED H. KHUDOR
AND BASIM M. HANOON
ﻤﻨﻻﺍ ﻥﻴﺒ ﺔﻗﻼﻌﻟﺍ ﺔﺴﺍﺭﺩ ﺩﺩﻌﺘ ﻊﻤ ﺔﻴﺌﺍﻭﺩﻟﺍ ﺔﻴﺴﺎﺴﺤﻟﺍﻭ ﺔﻴﻠﺼﻤﻟﺍﻭ ﺔﻴﻭﻴﺤﻟﺍ ﻁﺎ ﻥﻴﺠﻠﻟ ﻰﻨﻴﺠﻟﺍ لﺎﻜﺸﻻﺍCoa ﺔﻤﻭﺜﺭﺠ ﻰﻓ staphylococcus ﻥﻤ ﺔﻟﻭﺯﻌﻤﻟﺍ ﺭﺎﻘﺒﻻﺍ
ﺔﻁﺴﺍﻭﺒ
ﻥﻭﻨﺤ
ﺩﻤﺤﻤ
ﻡﺴﺎﺒﻭ
ﺭﻀﺨ
ﻥﺴﺤ
ﺩﻤﺤﻤ
،ﺱﺎﺒﻋ
ﺓﺭﻫﺯﻟﺍ
ﺩﺒﻋ
لﺴﺎﺒ
ﺕﺴﺭﺩ٤٠ ﺕﻻﻼﺴ ﻥﻤ ﺔﻔﻠﺘﺨﻤ ﻁﺎﻤﻨﺍ ﻥﻤ ﺔﻟﺯﻋ staphylococcus aureus ﻥـﻤ ﺔﻟﻭﺯﻌﻤﻟﺍ ﺭﺎﻘﺒﻻﺍ . ﻥﻴﺠ ﺩﻭﺠﻭ ﻥﻋ ﻯﺭﺤﺘﻟﺍ ﻡﺘcoa ﺔﻴﻨﻘﺘ ﻡﺍﺩﺨﺘﺴﺄﺒ RCR . ﺩﺩـﻌﺘ ﻥﻴﺠﻟﺍ ﺍﺫﻫ ﺭﻬﻅﺍ ﺩﻗﻭ ﻥﻴـﺒ ﺕـﺤﻭﺍﺭﺘ ﺔﻔﻠﺘﺨﻤ ﻡﺎﺠﺤﺄﺒﻭ ﺢﻀﺍﻭ لﻜﺸﺒ لﺎﻜﺸﺍ٥٠٠ ﻭ٦٠٠ﻭ٦٥٠ﻭ٨٠٠ ﻭ ٨٥٠ ﺝﻭﺯ ﻯﺩﻋﺎﻗ. ﺔـﻤﻭﺜﺭﺠﻟﺍ ﻩﺫﻬﻟ ﺔﻴﻠﺼﻤﻟﺍﻭ ﺔﻴﻭﻴﺤﻟﺍ ﻭ ﺔﻴﻨﻴﺠﻟﺍ ﻁﺎﻤﻨﻻﺍ ﻥﻴﺒ ﺔﻗﻼﻌﻟﺍ ﺔﺴﺍﺭﺩ ﻡﺘ ﺩﻗﻭ . ﺩـﻘﻟ ﺜﻜﻻﺍ ﺩﺠﺍﻭﺘﻟﺍ ﻥﺍ ﺩﺠﻭ ﻰﻠﺼﻤﻟﺍ ﻁﻤﻨﻠﻟ ﺭIV ﻱﻭﻴﺤﻟﺍ ﻁﻤﻨﻟﺍ ﻰﻓ C . ﻁﺎـﻤﻨﻼﻟ ﺩـﺠﺍﻭﺘ لﺠﺴﻴ ﻡﻟﻭ ﺔﻴﻠﺼﻤﻟﺍI ﻭ II ﻱﻭﻴﺤﻟﺍ ﻁﻤﻨﻟﺍ ﻰﻓ A . ﻰﻠﺼـﻤ ﻁﻨﻠﻟ ﺩﺠﺍﻭﺘ لﺠﺴﻴ ﻡﻟ ﺎﻤﻜV ﻥـﻤ ﻯﺍ ﻰـﻓ ﺕﻻﺯﻌﻟﺍ. ﻯﻭـﻴﺤﻟﺍ ﻁﻤﻨ ﻥﻤ ﺕﻻﺯﻌﻟﺍ ﻥﺍ ﺩﺠﻭ ﺩﻘﻓ ﻰﻨﻴﺠﻟﺍ ﻁﻤﻨﻟﺍ ﻊﻤ ﺔﻗﻼﻌﻟﺍ ﺹﺨﻴ ﺎﻤ ﺎﻤﺍA ﻭ C ﻡﺠﺤﺒ ﻰﻨﻴﺠﻟﺍ ﻁﻤﻨﻟﺍ ﺱﻔﻨ ﺎﻬﻟ ٥٠٠ ﻭ ٨٠٠ ﺝﻭﺯ ﻥﻴﺠﻟﺍ ﻥﻤ ﻯﺩﻋﺎﻗcoa ﺱـﻔﻨ ﻰـﻓ ﻊﻘﺘﻭ ﻰﻠﺼﻤﻟﺍ ﻁﺨﻟﺍIIﻭ IV. ﻥﻴﺠﻟﺍ ﻡﺠﺤﻭ ﺩﺠﺍﻭﺘ ﻥﻴﺒ ﺔﻗﻼﻌﻟﺍ ﺔﺴﺍﺭﺩ ﻥﻋ ﺎﻤﺍ coa ﺔـﻤﻭﺎﻘﻤﻟﺍ ﻊـﻤ ﻥﺍ ﺩﺠﻭ ﺩﻘﻓ ﻥﻴﺴﻴﺎﻤﻭﻜﻨﺎﻔﻟﺍﻭ ﻥﻴﻠﻴﺴﺎﺴﻜﻭﻼﻟ٥٠ % ﻥﻴﻠﻴﺴﺎﺴـﻜﻭﻼﻟ لـﻤﺎﻜﻟﺎﺒ ﺔﻤﻭﺎﻘﻤ ﺕﻨﺎﻜ ﺎﻬﻨﻤ ﻡﺠﺤﺒ ﻥﻴﺠﻟﺍ ﻥﺎﻜ ﺎﻤﺩﻨﻋ ﺔﻤﻭﺎﻘﻤ ﻰﻠﻋﺍ ﺕﺩﺠﻭﻭ٨٥٠ ﺒ ﻥﻴﺠﻟﺍ ﻪﻴﻠﻴ ﻯﺩﻋﺎﻗ ﺝﻭﺯ ﻡﺠﺤ٥٠٠ ﺝﻭﺯ ﻯﺩﻋﺎﻗ . ﻰﻓ ﺔﻤﻭﺎﻘﻤﻟﺍ ﺕﺩﺠﻭﻓ ﻥﻴﺴﻴﺎﻤﻭﻜﻨﺎﻔﻠﻟ ﺔﻤﻭﺎﻘﻤﻟﺍ ﺹﺨﻴ ﺎﻤﺍ٤٠ ﺔﻤﺎﺘ ﺔﻤﻭﺎﻘﻤﻟﺍ ﺕﻨﺎﻜ ﺔﻟﺯﻋ ﻰﻓ٥٠ % ﻡﺠﺤﺒ ﻥﻴﺠﻟﺍ ﻥﺎﻜ ﺎﻤﺩﻨﻋ ﺕﻨﺎﻜ ﺔﻤﻭﺎﻘﻤ ﻰﻠﻋﺍ ﻥﺍ ﹰﺎﻤﻠﻋ٨٥٠ﻯﺩﻋﺎﻗ ﺝﻭﺯ .

17 Sci. Cong. 2016, Fac. Vet. Med., Assiut Univ., Egypt
34
SUMMARY
Forty Staphylococcus aureus strains of different biotypes and serotypes,
previously isolated from bovine, were used in this study. The presence of
coagulase gen (coa) were investigated using PCR assay. coa gene shows
polymorphism since it appear in 500, 600, 650, 800 and 850 bp. The
relation between genotype, biotypes and serotypes of S. aureus were also
investigated. The relation between biotype and serotype of S. aureus
isolates revealed that the high occurrence of serotype IV was recorded in
the biotype C. No serotype I or II were recorded in biotype A isolates.
Serotype V not recorded in any strain. The relation between the biotype,
genotype and serotype in the same forty S. aureus isolates, showed that
the isolates from biotypes C and A, revealed that the same product of coa
gene (500 and 800 bp) share in the same serotype, Π and IV. The study
of the relation between the coa product with oxacillin showed that the
total resistance in genotypes to oxacillin was 50%. The high percentage
of resistance was found in isolates with coa product 850 bp followed by
500 bp. The relations appeared between the coa product with
vancomycine resistance in 40 bovine isolates were also recoded. The
total resistance in genotypes to vancomycine was 50%. The high
percentage of resistances was found in isolates with coa gene product 850
bp.
INTRODUCTION
Staphylococcus aureus acts as opportunistic pathogens and cause
infections of urinary tract, respiratory tract, and intestinal tract
(Olorunfemie et al., 2005). S. aureus is considered as pathogenic bacteria
when appearing in suitable condition. It is able to infect any part of the
body causing some diseases in human and animal that start with skin
infection, end to food poisoning and brain abscesses with outbreak in the
post operative wound infection (Kenneth, 2008). In the cows, it causes
some important diseases for example mastitis (clinical and sub clinical)
and respiratory tract infection, skin sepsis, Tic pyemia in lamb and
contagious skin necrosis (Kinight, 1999). The phenotypical
characterization of S. aureus biotypes establishes the presumptive origin
of isolation (Ordonez et al., 2005). Biotype has been stated that biotypes
S. aureus strains may give an indication of the origin contamination in
food products as the biotype correlates well with the animal host
(Lamperll et al., 2004).

17 Sci. Cong. 2016, Fac. Vet. Med., Assiut Univ., Egypt
35
S. aureus serotypng is epidemic indicator that depend on molecular
immunity possess organism and ability of B-lymphocyte to produce
antibody, this typing standing and stabile of epidemic indicator but it
does not have properties that be handled to differently. Some serological
method agglutination test, complement fixation tests,
immunoelectrophresis, radioimmuno assay are also found. The coagulase
protein is an important phenotypic determinant and accepted as a major
virulence factor of S. aureus. Analysis of coagulase encoding S. aureus
DNA coa gene has demonstrated variable sequences in the 3´ end coding
region (Goh et al., 1992; Kawaguchiya et al., 2013). This region contains
a polymorphism repeat region that can be used to differentiate S. aureus
isolates. This characteristic has been used to type S. aureus isolates of
human and bovine origin (Guler et al., 2005). The coa gene
polymorphism was used for typing and differentiation of S. aureus strains
isolated from bovine mastitic milk sample but using internal transcribed
spacer-PCR proved to be a useful and inexpensive procedure for
conducting epidemiological surveys of S. aureus (Ciftci et al., 2009;
Karakulska et al., 2011; Silveira-Filh et al.,, 2014) .
MATERIALS AND METHODS
Bacterial Strain: Forty S. aureus previously isolated from bovine were
used through this study.
Biotyping and serotyping: Biotyping of S aureus isolates were
determined according to Isigidi et al., (1990). All isolates were biotyped
by using pigment production, haemolysin, coagulation of bovine plasma
and growth on crystal violate. All isolates were serotyped to I, Π, ΠI, IV,
V, VI, VΠ, VΠI according to coagulase serotyping method.
Antibiotic susceptibility test : This test was done according to method
of Kirby and Bauer, (1966) using disc diffusion method.
PCR analysis: The coa gene was studied according to protocol of
(Hookey et al., 1998). Oligonucleotide primers sequences used for PCR
amplification of coa gene as follow:
Coa1 5´-ATA GAG ATG CTG GTA CAG G-3΄.
Coa2 5΄-GCT TCC GAT TGT TCG ATG C-3΄.

17 Sci. Cong. 2016, Fac. Vet. Med., Assiut Univ., Egypt
36
RESULTS
Biotyping and Serotyping of S. aureus
The results of biotyping of forty S. aureus isolates revealed that 83.33%
(34/40) were biotype C and 16.66% (6/40) were biotype A. There were
significant differences (P < 0.05) between biotype C and A of S. aureus
isolates. Coagulase enzyme used for serotyping of the same forty
isolates. The results obtained showed that the serotype IV had the high
occurrence (9 isolates) followed by VI and VΠ serotypes (6 isolates for
each). There were significant differences (P < 0.05) between serotypes in
bovine isolates. The relation between biotype and serotype of S. aureus
isolates revealed that the high occurrence of serotype IV was recorded in
the biotype C. No serotype I or II were recorded in biotype A isolates.
Serotype V not recorded in any strain (Table 1).
Molecular genetics study results (PCR on coa gene)
The results of DNA amplification of coa gene in S. aureus isolates
revealed that PCR products were found in 38 isolates at molecular size
500, 600, 650, 800, and 850 bp in percentage22.5%, 12.5%, 15%, 25%,
and 25% Respectively. There were no significant differences (p > 0.05)
between different coa gene products in bovine isolates (table 1). All
isolates with single band except two isolates showed 500 & 600 bp bands
(Fig. 1).
Relationship between biotypes, genotypes and serotypes
The relation between the biotype, genotype and serotype in the same
forty S. aureus isolates, showed that the isolates from biotypes C and A,
revealed that the same product of coa gene (500 and 800 bp) share in the
same serotype, Π and IV (Table 1).
Antibiotic susceptibility of S. aureus
The sensitivity results of forty isolates to thirteen antibiotics discs were
studied. Results showed susceptibility to tobramycin 65%, ciprofloxacin
62.5%, chloramphenicol 60%, clindamycin 52.5%, oxacillin 52.5% and
vancomycin 52.5%. The less susceptible results were showed to
erythromycin, gentamycin, nitrofurantoin, streptomycin, tetracycline, and
penicillin 45%-12.5%, as well as resistance to cloxacillin, that had
significantly differences (P < 0.01) between different antibiotics to
susceptibility of S. aureus isolates (Figure 2).
Relationship between biotypes, serotypes and ORSA and VRSA
The relationship between biotype A and C with ORSA and VRSA in S.
aureus isolates revealed high resistance of biotype C to oxacillin and less

17 Sci. Cong. 2016, Fac. Vet. Med., Assiut Univ., Egypt
37
to vancomycin, while in the biotype A showed high resistance to
vancomycin compared to oxacillin (Table 2).
The results also showed high resistance of serotypes to oxacillin were
recorded in serotype IV and VΠ, while the high resistance to
vancomycine recorded in the serotype VΠ also (Table 2).
Relationships between coa gene polymorphism and and resistance to
oxacillin (ORSA)and vancomycine (VRSA):
The study of the relation between the coa product with oxacillin
resistance in the 40 bovine isolates showed that the total resistance in
genotypes to oxacillin was 50%. The high percentage of resistance was
found in isolates with coa product 850 bp followed by 500 bp The
relations appeared between the coa product with vancomycine resistance
in 40 bovine isolates were also recoded. The total resistance in genotypes
to vancomycine was 50%. The high percentage of resistances was found
in isolates with coa gene product 850 bp (Table 3).
DISCUSSION
In this study the results showed the specific biotype C to bovine isolates
predominant in bovine isolates. Bendahou et al., (2008) showed that the
four biotypes A, B, C and unspecific of the S. aureus isolates from milk
and milk production appeared bovine origin biotype C recorded and more
dominant than the other biotypes. In the present study the bovine isolates
showed high percentage of recorded in serotype IV, VII ,VI and Π.
Many other studies on serotyping of animals isolates were carried out.
Hata et al., (2006) tested 231 S. aureus isolates (161clinical mastitis and
70subclinical mastitis) collect from 214 farms. Moon et al., (2007) who
found 4 main serotypes comprising I ,VIII , II and VII when used ten
coagulase serotype patterns on S. aureus isolates. The isolates of S.
aureus from raw milk sample for coagulase serotyping revealed, serotype
Π , ΠI ,IV, VΠ, VΠI recorded while the serotype I, V, VI was not
recoded (Pork et al., 2001). The serotypes were recorded similar in
biotype A isolates were Π, IV, VI VΠ, VΠI, while the serotypes recorded
similar in biotype C isolates were Π ΠI, IV VI. In the two aforesaid
studies revealed the serotype recorded in the animals able to record in the
human and vice versa. Coagulase serotyping method has been used
successfully in the epidemiological investigation staphylococcal food
poisoning, coagulase serotypes VII, accounted for about 70% of the total
outbreak. Coagulase serotypes III, 12%, serotypes II, 11% and serotypes

17 Sci. Cong. 2016, Fac. Vet. Med., Assiut Univ., Egypt
38
VI, 3%, of coagulase responsible for food poisoning incidence (Shimizu
et al., 2000).
The results of this study on coa gene revealed polymorphism phenomena
in this gene resulted of different molecular weight 500 bp, 600 bp, 650 bp,
800 bp and 850 bp. Testing for multiple gene polymorphisms is more
useful for local epidemiologic studies (Omer et al., 2014). In the bovine
isolates the present study recorded 500 bp, 600 bp, 650 bp, 800 bp and
850 bp of coa gene from S. aureus which isolated from different tested
samples. This region contains a polymorphism repeat region that can be
used to differentiate S. aureus isolates. This characteristic has been used
to type S. aureus isolates of human and bovine origin (Guler et al., 2005;
Hooky et al., 1998). Other study showed that S. aureus isolates of bovine
mastitic milk origin from herds at different locations were revealed three
different types of coa gene products 400bp, 510bp, 600 bp, 680 bp,
700bp and 850 bp (Salasia et al., 2004; Maslankova et al., 2009; Coelho
et al., 2009).
In this study in bovine isolates the total resistance to oxacillin and
vancomycine were 50%. The antibiotics resistance pattern revealed
resistance to oxacillin and vancomycin in 25% and 17% respectively
(Coelho et al., 2009). In the study of (Ordonez et al., 2005), the
percentage of resistance of S. aureus isolated from mastitis is 19%. The S.
aureus isolated from different patients from clinical specimens the
molecular typing of MRSA appeared 54 isolates with 64.28%, while
MSSA recorded 30 isolates with 35.71%, by used restriction digestion of
coa PCR product (Tiwari et al., 2008). Enterotoxin genes were more
prevalent in MRSA than in MSSA and the MSSA strains were
genetically more diverse than the MRSA strains (Lim et al., 2012).
The relationship between isolates origin with genotype and serotype,
bovine biotypes C and A, were appeared the same product of coa gene
(500 and 800 bp) share in the same serotype, Π, IV. Six hundred nightly
six isolates of S. aureus isolated from milk mastitis cows. The test of coa
gene polymorphism showed ranging between 620-809 bp for each S.
aureus. The serotype recoded serotype I 25.4% followed by VΠI 17.8%,
serotype Π 13.9% and serotype VΠ 13.2% (Moon et al., 2007). The
coagulase serotypes showed serotype I 1.46%, serotype Π 64.30%,
serotype ΠI 5%, serotype IV 7.37, serotype V 3.68%, serotype VI 0.44%,
serotype VΠ 13.27% and serotype VΠI 1.91% while recorded non
specific 2.5%. The coa gene polymorphism recoded different products
350 bp, 431 bp, 512 bp, 593 bp, 674 bp, 755 bp, 836 bp and 917 bp, in
the isolates from nosocomal infection in human (Ishino et al.,

17 Sci. Cong. 2016, Fac. Vet. Med., Assiut Univ., Egypt
39
2007,). Amplification of the coagulase gene from 86 S. aureus strains
isolates by specific primers showed 31 specimens contained 970 bp
fragment, and 11 strains contained 730 bp fragment relevant to coa gene
(coagulase) in PCR (Momtaz et al., 2011).
CONCLUSION
The relation between S. aureus biotype, genotype and serotype showed
that the isolates from biotypes C and A have the same product of coa
gene (500 and 800 bp) and found in the same serotype. coa gene shows
polymorphism since it appear in different size. Fifty percent of isolates
having coa gene showed resistance to oxacillin and vancomycine.
Table (1): Relationship between biotype, coa gene product and
serotype in the bovine isolates
Coa
bp Iisolates
No (%) Biotype
C Serotype Biotype
A Serotype
500 9(22.5) 7 I, (Π) 2, (ΠI) 2, IV, VI 2 Π, IV
600 5 (12.5) 5 (VI) 2, Π, ΠI, IV. 0 /
650 6(15) 4 Π, VI, (IV)2 2 VΠ, VΠI
800 10(25) 8 I, Π,( ΠI)2, VI, IV,
(VΠ)2 2 Π, IV
850 10(25) 10 (I)2 ,( IV) 2, VI,
V( Π)3, ( VΠI)2 / /
Total 40 34 I=4, Π=5, ΠI=5,
IV=7, VI=6,
VΠ=5, VΠI=2
6 Π=2, IV=2,
VΠ=1, VΠI=1

17 Sci. Cong. 2016, Fac. Vet. Med., Assiut Univ., Egypt
40
Table (2): Relationships between serotypes and resistance to ORSA and VRSA of S.
aureus
Serotype Milk Vaginal
swabs Nasal
Swabs Total (%)VRSA
(%)ORSA
I 1 1 2 4 2 (50) 2 (50)
Π 5 1 0 6 3 (50) 1 (33.3)
IΠ 3 1 0 4 1 (25) 1 (25)
IV 6 2 2 10 7 7(70)
V 0 0 0 0 0(0.0) 0(0.0)
VI 3 2 1 6 3(50) 3(50)
VΠ 2 2 3 7 4(57.1) 4(57.1)
VΠI 0 1 2 3 1(33.3) 1(33.3)
Total 20 10 10 40 18(45) 19(47.5)
Biotype
A 4 2 - 6 3(50) 2(33.33)
C 16 8 10 34 16(47) 17(50)
Total 20 10 10 40 19(47.5) 19(47.5)
Table (3): Relationships between ORSA, VRSA and coa gene polymorphism of S.
aureus isolates
Sample/No.
Resistance
500
bp 600
Bp 650
bp 800
bp 850
bp %
Resistance
Milk /20 ORSA
VRSA
6
6
0
0
0
1
4
5
0
0
50
55
Vaginal
swabs/10 ORSA
VRSA 0
0 1
1 2
1 0
0 0
0 30
20
Nasal
swabs/10
ORSA
VRSA 0
0 0
0 0
0 0
0 7
6 70
60
Total / (%) ORSA
VRSA
6(15)
6(15)
1(2.5)
1(2.5)
2(5)
2(5)
4(10)
5(12.5)
7(17.5)
6(15)
50
50

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41
Fig. (1) Electrophoresis of coa gene PCR products in 1% agarose. A; Lane 1=
100bp DNA ladder, 2&3= 500bp, 4= 650bp, 5&6= 600bp, 7&8 =850bp. B; Lane 1=
100bp DNA ladder, 2&3= 500bp, 4-7= 650bp, 8= 800bp. C; Lane 1= 100bp DNA
ladder, 2= double band of 500 & 650 bp.
Fig. 2: The percentage of antibiotic susceptibility of Staphylococcus aureus agaivst
different antibiotics

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