antibiotic resistance of the enterococcal isolates...

CHAPTER 6

ANTIBIOTIC RESISTANCE OF THE ENTEROCOCCAL ISOLATES

6.1. INTRODUCTION

Antibiotics are extensively used to prevent or to treat microbial

infections in human and veterinary medicine. Steadily increasing antibiotic

resistance and decreasing numbers of newer antibiotics has become a global

problem. Over the last two decades, nosocomial infections caused by

Enterococcus particularly VRE have emerged and have subsequently been

isolated from all over the world. (Rice, 2000; Oteo et al., 2007; Top et al.,

2008; Suchitra and Laksmidevi, 2009). The emergence of E. faecalis and E.

faecium was paralleled by the increase in glycopeptide and high-level

aminoglycoside resistance which are important drugs for the treatment of

gram positive bacterial infections (Shepard and Gilmore, 2002).

To date six gene clusters like vanA, vanB, vanC, vanD, vanE and

vanG (Boyd et al., 2006) have been found to be associated with vancomycin

resistance in Enterococcus species. It is very likely that this resistance could

be horizontally transferred to other organisms (Arthur and Courvalin, 1993).

The epidemiology of vancomycin resistant E.faecium is yet to be well

understood. There is difference in opinion about the emergence of

vancomycin resistance. Nosocomial spread of VREF has been reported by

Bonten et al., (2001). Others revealed that commensal microbiota of some

animals and humans act as reservoirs of VREF (Jensen et al., 1998; Klare et

al., 1995). VREF of food borne origin is also known to cause human

colonization (Bogaard et al., 1996). However the role played by non human

124 Chapter 6

sources and reservoirs other than hospitalized patients in the spread of

Enterococcus is ambiguous (Turnidge, 2004). Many molecular techniques

are used for studying the genetic relatedness of Enterococcus. The

effectiveness of RAPD in enterococcus typing was described by Barbier et

al., (1996).

Concern over increasing resistance to antimicrobial agents has

highlighted the need for surveillance of antimicrobial resistance at a local,

national and international level to tackle this problem. In this study, a

population of Enterococcus species from various sources was examined for

the occurrence of resistance against antimicrobials employed in veterinary

and in human medicine and also to compare antimicrobial sensitivity against

Enterococcus isolated from different sources. In the study, RAPD analysis

was conducted to compare the genetic similarities of VREF isolates from

clinical and poultry sources.

6.2. MATERIALS AND METHODS

Different enterococcal strains obtained from clinical and nonclinical

isolates especially from water, chicken faeces and human faeces were

screened for antibiotic resistance.

6.2.1. Antimicrobial susceptibility of Enterococcus

Antimicrobial susceptibility of the isolate was tested by the disc

diffusion method according to Bauer et al., (1966). Bacterial cultures were

prepared from overnight growth on a blood agar plate by suspending seven

to eight morphologically similar colonies in peptone water. Each inoculum

was adjusted to 0.5 Mc Farland standards. The inoculum was swabbed on to

Mueller -Hinton Agar. Commercially prepared antibiotic discs obtained from

Hi-Media (Appendix-2) with a diameter of 6 mm were used to determine the

Antibiotic Resistance of the Enterococcal Isolates 125

susceptibility pattern of Enterococcus species. The plates were incubated at

37o C over night. Zone of inhibition was measured and the results were

interpreted according to the National Committee for Clinical Laboratory

Standards, (2003).

6.2.2. MIC determination

E-test antibiotic strips (HiMedia) were used to determine the

susceptibility of the strains to ampicillin, benzyl penicillin, streptomycin,

gentamicin, vancomycin and teicoplanin. Mueller–Hinton agar plates were

lawn-cultured with enterococci, and antibiotic strips were placed onto the

plates according to the manufacturer's instructions. Plates were incubated at

37°C, and results were read after 24 and 48 h of incubation. The MIC values

were determined from the concentration of antibiotic at which the zone

intersects the test strip (Huang et al., 1992)

6.2.3. Beta lactamase test:

Production of beta lactamase was determined by using cefinase discs

(BD Diagnostic Systems) which were impregnated with chromogenic

cephalosporin, nitrocefin. Colonies were smeared onto the surface of

moistened disc and observed for colour change (Montgomery et al., 1979).

6.2.4. PCR amplification of vancomycin resistance gene

Three vancomycin resistant isolates (1 vanA & 2vanB phenotypes)

were selected for the detection of resistance gene determinants by

Polymerase Chain Reaction (PCR). The selected isolates were subjected to

both genomic DNA isolation and plasmid purification.

126 Chapter 6

6.2.4.1. Genomic DNA Isolation

Genomic DNA isolation was carried out as per the methods

explained by Kitao et al., (2010). For this, 1.2×104 CFU of the pure cultured

bacteria were mixed with 0.5mL IM NaCl supplemented 10 mM Tris HCl

buffer (pH 8.0) in a microtube. This was followed by the addition of 5µl

(1,000 U/mL) lysostaphin (Sigma – Aldrich) and incubated at 37°C for 1 h.

The sample was further kept for 15min on a boiling water bath and was then

centrifuged at 12,000 rpm for 10 min. The concentration of DNA in the

supernatant was measured at 260nm, and was further diluted to 100ng/µl

concentration for the PCR.

6.2.4.2. Plasmid DNA Isolation

Plasmid DNA was isolated by alkali lysis method as described by

Birnboim and Doly, (1979) with slight modification. For this, a test tube

containing 5mL of brain heart infusion broth was inoculated with a single

isolated colony picked from BHIA and this was incubated at 37°C, overnight

with shaking. From this, 1.5mL of culture was taken in a microfuge tube and

was centrifuged for 30 seconds at 12,000g at 4°C. The supernatant was

discarded and the bacterial pellet was resuspended in 100µl of solution I

(Appendix-3) and kept for 5 minutes in ice. This was followed by the

addition of 200µl of solution II (Appendix-3) and was mixed by inverting

the tube gently. The tube was stored on ice for 10 minutes. Then 150µl of

ice cold solution III (Appendix-3) was added and mixed by inverting the

tube gently. The tube was then kept for 15 minutes on ice to form white

precipitate. The tube was again centrifuged for 10 minutes at 14,000 rpm.

The supernatant was transferred to a fresh tube without transferring any of

the white precipitate. This was followed by addition of two volumes of


ethanol to the supernatant to precipitate the plasmid DNA. This was then

mixed well by inverting the tube several times and the mixture was allowed

to stand for 30 minutes on ice. The precipitated plasmid was collected by

centrifuging at 14,000 rpm for 10min. The supernatant was removed and

discarded and to the pellet 1mL of ice cold 70% ethanol was added and

centrifuged again for 30 seconds. The supernatant was removed and

discarded. The pellet was air dried for 10-30 minutes and then it was

resuspended in 50µl of sterile distilled water and stored at -20°C. The

quality of the plasmid DNA was checked by electrophoresis using 1.2%

agarose gel.

Vancomycin resistant gene was amplified by PCR from genomic

DNA and plasmid DNA using vancomycin specific primers. Three

vancomycin resistant E.faecium genomic DNA were initially screened for

vanA and vanB by PCR. In those cases, where PCR was negative again

subjected to PCR using plasmid DNA as template. The primers used for Van

A gene amplification was VanAF (5’- GGGAAAACGACAATTGC-3’) and

VanAR (5’-GTACAATGCGGCCGTTA-3’). The primers for Van B gene

amplification were VanBF (5’-ACCTACCCTGTCTTTGTGAA-3’) and

VanBR (5’-AATGTCTGCTGGAACGATA-3’) respectively. The primer

sequences were got synthesized from Sigma – Genosys. PCR was carried

out in a final volume of 50ul with 50 ng of genomic DNA/plasmid DNA, 20

pmol of each primer, 200 µM of each dNTPs, 5 µl of 10X PCR buffer and

1.25 U of Taq DNA polymerase in a Mycycler™ (Bio-Rad, USA). The PCR

conditions used were; 5 minutes denaturation at 94°C, followed by 30 cycles

of 1 min denaturation at 94°C, 1 min annealing at 50°C , 1 min extension at

72°C, and a final extension for 7 minutes at 72°C. PCR products were

128 Chapter 6

visualized by 1.5% (w/v) agarose gel. This part of the work was carried out

at Unibiosys lab, Cochin

6.2.4.3. Sequencing and analysis

The PCR product was further gel purified and subjected to DNA

sequencing. This part of the work was carried out at Sci Genome, Cochin.

The sequence data of both vanA and vanB genes were then subjected to

BLAST analysis. The data was also used for multiple sequence alignment

and phylogenetic analysis with other sequences from database.

6.2.5. Resistance gene transfer by conjugation

In order to confirm the transfer of plasmid carrying vancomycin

resistance (VanB) to other enterococci, conjugation studies were conducted.

E.faecium with vanB plasmid was selected as donors based on their

resistance to vancomycin (vanr) and sensitivity to rifampicin (Rif s). This

vanr Rif s strain was mixed with E.faecium which were sensitive to

vancomycin and resistant to rifampicin. Broth mating was done as described

by Ike et al., (1998) with modifications. Here, 0.5 mL of donor culture was

mixed with 0.5 mL of the recipient culture and was added to 5 mL of Luria

Bertani broth (Appendix 3). The mixture was incubated at 37° C for 4 hours

with gentle agitation for the appropriate time. Then the transconjugants were

selected on agar plates containing 16µg/mL vancomycin and 20 µg/mL of

rifampicin. Colonies were counted after 48 h of incubation at 37°C. The

conjugation was confirmed by their antibiotics resistance patterns and

plasmid detection. The conjugation efficiency was determined based on the

number of transconjugants per donor cells.


6.2.6. Random amplified polymorphic DNA analysis

RAPD analysis was carried out to investigate role of clonal spread of

VREF, in the emergence of van B type of VREF. By detecting and

sequencing the resistance genes, similar plasmid borne vanB type were

detected and to rule out the clonal spread of VREF, RAPD was carried out.

One vanB type of vancomycin resistant enterococci from chicken source and

a vanB type of vancomycin resistant enterococci from blood were randomly

selected for molecular typing by RAPD test. The sequence of the random

primer used for DNA amplification were OPK7: AGCGAGCAAG, OPK11:

AATGCCCCAG, OPK12: TGGCCCTCAC, OPBG 19: GGTCTCGCTC,

OPE6: AAGACCCCTC, OPL7. AGGCGGGAAC, OPAA14:

AACGGGCCAA, OPAA 17: GAGCCCGACT. Each reaction mixture (25

µl) contained template DNA (20-25 ng), 10X Taq buffer , MgCl2 (25mM),

dNTP mix (10mM), Primer (10pmol) Taq DNA polymerase (0.3U). The

thermo cycler was programmed with an initial denaturation at 94° C for

3minutes followed by cyclic denaturation at 94°c for 30cycles for 45

seconds, annealing at 37° C for 1 minute and extension at 72° C for 1

minute. The PCR was conducted for 40 cycles. Finally it was subjected to

extended polymerization at 72° C for 6 minutes. Approximately 10

microliter PCR products were electrophoresed on 2% (w/v) agarose gel

prepared in 1X TBE Buffer at 80 V for 2 h. This was followed by staining

with ethidium bromide (0.5 µg/mL).

6.3. STATISTICAL ANALYSIS

Significance in percentage difference is calculated by chi square test.

When the Chi square tests were not valid Yates correction were applied and

Fisheres exact test were also done where ever necessary. Statistical analysis

130 Chapter 6

of MIC of antibiotics was carried out by ANOVA. Analysis was done by

using SigmaStat software (Sigma-Aldrich, St. Louis USA). The level of

significance was set up at P <0.05.

6.4. RESULTS

Many of the isolates were resistant to multiple antibiotics. All

E.faecium and E.faecalis isolates tested showed resistance to clindamycin

and all remained susceptible to linezolid. Penicillin resistance was high in the

isolates.

The resistance to different antibiotics shown by the isolates of

different Enterococcus from water is shown in Table 6.1.

Table 6.1.Antibiotic resistance in isolates of Enterococcus from water

Antibiotics

No.&% *of resistant isolates

E.faecium

(n=100)

E.faecalis

(n=56)

E.raffinosus

(n=10)

E.avium

(n=10)

E.durans

(n=10)

E.gallinarum

(n=14)

No. & % No. &% No.&% No.&% No.&% No. & %

Penicillin 100(100) 56(100) 10(100) 5(50) 5(50) 7(50)

Ampicillin 42(42) 46(82.1) 0 0 0 0

Oxacillin 70(70) 45(80.4) 2(20) 4(40) 2(20) 2(14.28)

Cindamycin 100(100) 56(100) 10(100) 5(50) 5(50) 7(50)

Linezolid 0 0 0 0 0 0

Gentamicin 42(42) 36(64.3) 0 0 0 0

Ciprofloxacin 14(14) 36(64.3) 0 0 0 0

Erythromycin 14(14) 36(64.3) 0 0 0 0

Chloramphenicol 20(20) 8(14.3) 0 0 0 0

Tetracycline 14(14) 8(14.3) 10(100) 5(50) 5(50) 7(50)

Amoxyclav 0 8(14.3) 0 0 0 0

Teicoplanin 0 0 0 0 0 0

Gentamicin 120 0 2(3.6) 0 0 0 0

Vancomycin 0 0 0 0 0 0

*: percentage of isolates is given in brackets


Penicillin resistance was significantly high in water isolates

(p<0.001). None had linezolid, teicoplanin and vancomycin resistance.

Amoxyclav resistance shown by few E.faecalis isolates. Ampicillin

resistance was high in the isolates.

The resistance to different antibiotics shown by the clinical isolates of

enterococci is summarized in Table 6.2.

Table 6. 2. Antibiotic resistance in isolates of Enterococcus from clinical sources

Antibiotic

No and percentage* of resistant isolates

E.faecalis

(n=148)

E.faecium

(n=52)

E.gallinarum

(n=2)

E.raffinosus

(n=2)

E.avium

(n=1)

No.& % No.&% No.&% No.&% No.&%

Penicillin 120(81.1) 38(73.1) 1(50) 1(50) 1(100)

Ampicillin 20(13.5) 14(26.9) 1(50) 1(50) 1(100)

Gentamicin 104(70.3) 34(65.4) 0 0 0

Oxacillin 105(70.9) 30(57.7) 1(50) 1(50) 1(100)

Streptomycin 88(59.5) 18(34.6) 1(50) 0 0

Gentamicin 120 60(40.5) 32(61.5) 0 0 0

Amoxyclav 20(13.5) 14(26.9) 1(50) 0 1(100)

Tetracycline 54(36.5) 14(26.9) 1(50) 0 1(100)

Erythromycin 98(66.2) 28(53.8) 0 0 0

Ciprofloxacin 60(40.5) 18(34.6) 1(50) 0 0

Chloramphenicol 8(5.4) 6(11.5) 0 0 0

Nitrofurantoin 10(6.8) 10(19.2) 0 0 0

Clindamycin 148(100) 52(100) 2(100) 2(100) 1(100)

Vancomycin 0 6(11.5) 0 0 0

Teicoplanin 0 3(5.8) 0 0 0


132 Chapter 6

Majority of the isolates have shown resistance to most of the tested

antibiotics. Penicillin and oxacillin resistance was found to be high in the

isolates. Six of the E.faecium isolates were vancomycin resistant. Though

ampicillin resistance was less, aminoglycoside resistance was high.

Resistance to gentamicin (30 µg and 120 µg) and amoxyclav was distributed

with high frequency in clinical isolates (P<0.001).

The resistance to different antibiotics shown by different enterococci

isolates from chicken is summarized in Table 6.3.

Table 6.3. Antibiotic resistance in isolates of Enterococcus from chicken faeces

Antibiotic

No.and % *of resistant isolates

E.faecium

(n=130)

E.faecalis

(n=4)

E.gallinarum

(n=5)

E.avium

(n=5)

E.raffinosus

(n=10)

E.durans

(n=3)

No.&% No.&% No.&% No.&% No.&% No.&%

Penicillin 120(92.3) 3(75) 4(80) 4(80) 8(80) 2(66.7)

Ampicillin 9(6.9) 2(50) 2(40) 0 0 0

Oxacillin 20(15.4) 0 0 0 0 0

Gentamicin 13(10) 3(75) 1(20) 0 0 0

Gentamicin 120 0 0 0 0 0 0

Amoxyclav 9(6.9) 0 0 0 0 0

Tetracycline 100(76.9) 0 0 0 0 0

Erythromycin 115(88.5) 1(25) 0 0 0 0

Ciprofloxacin 23(17.7) 0 0 0 0 0

Clindamycin 130(100) 4(100) 5(100) 5(100) 10(100) 2(66.7)

Chloramphenicol 7(5.4) 2(50) 0 0 0 0

Linezolid 0 0 0 0 0 0

Nitrofurantoin 0 0 0 0 0 0

Teicoplanin 0 0 0 0 0 0

Vancomycin 20(15.4) 0 0 0 0 0



All the Enterococcal isolates from chicken isolates were resistant to

penicillin and had a high clindamycin resistance. A high resistance

percentage was obtained against erythromycin followed by tetracycline.

None were found to be resistant to linezolid and streptomycin (data not

shown). Gentamicin resistance was very low. Vancomycin resistance was

shown by 20 E.faecium isolates. Penicillin, tetracycline and erythromycin

resistance was significantly high in chicken isolates (P<0.05).

The antibiotic resistance pattern shown by human faecal isolates are

presented in table 6.4.

Table 6.4. Antibiotic resistance in isolates of Enterococcus from human faeces

Antibiotic

No.and % *of resistant isolates

E.faecium (n=52)

E.faecalis (n=2)

E.avium (n=12)

No.&% No.&% No.&%

Penicillin 32(61.5) 0(0) 0(0)

Ampicillin 10(19.2) 0(0) 0(0)

Oxacillin 12(23.1) 0(0) 0(0)

Gentamicin 16(30.8) 1(50) 1(8.3)

Gentamicin 120 4(7.7) 0(0) 0(0)

Amoxyclav 0(0) 0(0) 0(0)

Tetracycline 12(23.1) 0(0) 0(0)

Erythromycin 16(30.8) 0(0) 0(0)

Ciprofloxacin 8(15.4) 0(0) 0(0)

Clindamycin 52(100) 2(100) 11(91.7)

Chloramphenicol 8(15.4) 0(0) 0(0)

linezolid 0(0) 0(0) 0(0)

Nitrofurantoin 8(15.4) 0(0) 0(0)

Teicoplanin 0(0) 0(0) 0(0)

Vancomycin 0(0) 0(0) 0(0)

* % of isolates is given in brackets

134

The percentage

antibiotics was

nitrofurantoin 15%.

Resistance to different groups of antibiotics shown by the isolates from

clinical and environmental sources

Figure 6.1(a-m).

Among the beta lactam

more in the environmental isolates compared to the human.

Enterococcus were resistant to penicillin.

0

20

40

60

80

100

120

E.faecium

Pe

rce

nta

ge

The percentage incidence of resistance of E. faecium towards different

antibiotics was ampicillin 19%, gentamycin 31%, HLGR 8%

ntoin 15%.


clinical and environmental sources is compared in Figures 6. a-m

Occurrence of antibiotic resistance in isolatesEnterococcus from clinical and environmental

Figure 6.1.a. Resistance to penicillin

Among the beta lactam antibiotics tested, resistance to penicillin was

more in the environmental isolates compared to the human.

were resistant to penicillin.

E.faecium E.faecalis E.avium E.gallinarum

Human Environmental

Chapter 6

towards different

31%, HLGR 8% and


m.

of antibiotic resistance in isolates of clinical and environmental sources

antibiotics tested, resistance to penicillin was

All species of

E.raffinosus

Antibiotic Resistance of the Enterococcal Isolates

Figure

Ampicillin resistance in

sources. Ampicillin resistance in

environmental isolates compared to the human isolates.

E.avium, E.gallinarum

environmental isolates.

Figure

Oxacillin resistance was also found in both environmental and human

sources. Oxacillin resistance was seen chiefly in

0

20

40

60

80

100

E.faecium

Pe

rce

nta

ge

0

10

20

30

40

50

60

70

80

E.faecium

Pe

rce

nta

ge

he Enterococcal Isolates

ure 6.1.b. Resistance to ampicillin

Ampicillin resistance in E.faecium was low in isolates from both the

sources. Ampicillin resistance in E.faecalis isolates was higher in

environmental isolates compared to the human isolates. Human isolates of

and E.raffinosus were more resistant than

Figure 6.1.c. Resistance to oxacillin


Oxacillin resistance was seen chiefly in E.faecalis.

E.faecalis E.avium E.gallinarum E.raffinosus

Human Environmental


Human Environmental

135

was low in isolates from both the

isolates was higher in

n isolates of

were more resistant than


E.raffinosus

E.raffinosus

136

Amoxyclav

absent in the isolates of

E.raffinosus.

Figure

Gentamicin (low level) resistance was more in human

isolates. A large number of environmental isolates was also found to be

resistant. E.avium

also had this property.

0

10

20

30

40

50

60

Pe

rce

nta

ge

0

10

20

30

40

50

60

70

80

E.faecium

Pe

rce

nta

ge

Figure 6.1.d. Resistance to amoxyclav

moxyclav resistance was very less in all isolates and was totally

solates of E.avium, environmental isolates of E.gallinarum

Figure 6.1.e. Resistance to gentamicin(30µg)

Gentamicin (low level) resistance was more in human


E.avium human isolates and E.gallinarum environmental isolates

also had this property. Resistance was absent in E.raffinosus.

E.faecium E.faecalis E.avium E.gallinarum E.raffinosus

Human Environmental


Human Environmental

Chapter 6

and was totally

E.gallinarum and

Gentamicin (low level) resistance was more in human E.faecalis


environmental isolates

E.raffinosus

E.raffinosus


Figure 6.1

HLGR was predominantly seen

was not present in the isolates

Figure

Tetracycline resistance was more prevalent among environmental

isolates of E.faecium. E.faecalis

be more resistant than environmental isolates.

0

5

10

15

20

25

30

35

40

45

E.faecium

Pe

rce

nta

ge

0

10

20

30

40

50

60

E.faecium

Pe

rce

nta

ge


6.1.f. Resistance to gentamicin(120µg)

HLGR was predominantly seen E.faecalis of human origin.

isolates of E.avium, E.gallinarum and E.raffinosus

6.1.g. Resistance to tetracycline

resistance was more prevalent among environmental

E.faecalis isolates from human sources were found to

be more resistant than environmental isolates. Resistance was found in the


Human Environmental


Human Environmental

137

HLGR

E.raffinosus.

resistance was more prevalent among environmental

solates from human sources were found to

Resistance was found in the

E.raffinosus

E.raffinosus

138

human isolates of

the environmental isolates of

Figure

Human isolates of

Environmental E.faecium

isolates of E.faecium

Figure

Ciprofloxacin resistance was high in environmental isolates of

E.faecalis. E.faecium

0

10

20

30

40

50

60

70

E.faecium

Pe

rce

nta

ge

0

10

20

30

40

50

60

70

E.faecium

Pe

rce

nta

ge

isolates of E.raffinosus ,E.avium and E.gallinarum. It was absent in

the environmental isolates of E.raffinosus and E.gallinarum.

Figure 6.1.h.Resistance to erythromycin

Human isolates of E.faecalis had a high resistance to erythromycin.

E.faecium isolates exhibited more resistance than human

E.faecium. Other enterococci were found to be sensitive.

Figure 6.1.i. Resistance to ciprofloxacin


.faecium isolates and E.avium from human sources


Human Environmental


Human Environmental

Chapter 6

It was absent in

had a high resistance to erythromycin.

more resistance than human

Other enterococci were found to be sensitive.


from human sources were more

E.raffinosus

E.raffinosus


resistant than environmental isolates.

and all isolates of E.avium

Figure 6.1

Chloramphenicol

isolates of E.faecalis. E.faecium

chloramphenicol. All other enterococci species were sensitive.

Figure 6.1

Nitrofurantoin resistance was

E.faecium and E.faecalis.

0

5

10

15

20

E.faecium

Pe

rce

nta

ge

0

5

10

15

20

E.faecium

Pe

rce

nta

ge


resistant than environmental isolates. Environmental isolates of E.gallinarum

E.avium and E.raffinosus were sensitive to the drug.

6.1.j. Resistance to chloramphenicol

resistance was more frequently in environmental

E.faecium from human sources was more resistant

All other enterococci species were sensitive.

6.1.k. Resistance to nitrofurantoin

resistance was found in human isolates in the case of

E.faecalis. All other isolates tested were sensitive.


Human Environmental


Human Environmental

139

E.gallinarum

were sensitive to the drug.

in environmental

resistant to

in the case of

E.raffinosus

E.raffinosus

140

Teicoplanin resistance was found only in the human isolates of

E.faecium. All other

Figure

The vancomycin resistance was more in isolates fr

sources compared to human isolates.

E.gallinarum isolates

found only in the isolates of

0

0.5

1

1.5

2

2.5

3

3.5

E.faecium

0

2

4

6

8

10

E.faecium

Pe

rce

nta

ge

Figure 6.1.l. Resistance to teicoplanin


other isolates tested were sensitive to the drug.

Figure 6.1.m. Resistance to vancomycin

The vancomycin resistance was more in isolates from environmental

sources compared to human isolates. E.faecalis, E.avium, E.raffinosus

isolates tested were sensitive to vancomycin. Resistance was

found only in the isolates of E.faecium.

E.faecalis E.faecalis E.gallinarum E.raffinosus

Human Environmental

E.faecium E.faecalis E.faecalis E.gallinarum

Human Environmental

Chapter 6


m environmental

E.raffinosus and

Resistance was

E.raffinosus

E.raffinosus


The minimum inhibitory concentrations of the selected antibiotics

towards different Enterococcus species are shown in Table 6.5

Table 6. 5. Minimum Inhibitory Concentration of drugs against enterococci

Antibiotic

Range of MIC

E.faecalis (n=30)

E.faecium (n=30)

E.gallinarum (n=5)

E.raffinosus (n=5)

E.avium (n=5)

Penicillin 8 to 16 8 to 16 8 8 8to16

Streptomycin 0.1 to >240 0.1 to >240 0.1 0.5 16--32

Teicoplanin .01 to 0.05 0.05 to 64 0.01-4 0.01 0.01

Vancomycin 0.5 to 2 0.5 to 128 0.5-1 1 0.5

Gentamicin 0.128 - >1024 0.512 - >1024 0.128 0.512 0.128

Ampicillin 0.5 – 32 0.5 – 32 0.5 0.5 0.5

*No. of isolates tested is given in brackets

Though penicillin resistance was expressed by most of the strains, the

MIC was below 16µg/mL. Ampicillin MIC observed in this study was also

between 0.5 to 32µg/mL. HLGR and HLSR were distributed among the

isolates. Vancomycin MIC was significantly more in E.faecium than other

species (p<0.001). High-level resistance to vancomycin and teicoplanin

(MIC >128& 64 µg/mL) observed in this study corresponds to vanA

phenotype. Low level resistance to vancomycin (MIC >16-32 µg/mL) and

sensitivity to teicoplanin matches to vanB phenotype.

Antibiotic sensitivity pattern shown by Enterococcus is displayed in

Plates 6.1. Plate 6.2 shows the pattern of MIC towards different antibiotics

obtained by the E test.

142 Chapter 6

Plate.6.1: Antibiotic sensitivity test by disc diffusion method

Plate 6.2: E test for detection of MIC


Genomic DNA and plasmid DNA was isolated from selected

isolates. Plate 6.3(A) shows the gel pictures of DNA isolated from sample

VREF1 (clinical), VREF2 (clinical) & VREF4 (chicken) and plate 6.3. (B)

shows the gel picture of plasmids from VREF1&VREF4

Plate 6.3. Agarose gel electrophoresis of genomic DNA and Plasmid

DNA of VREF

(A) . Genomic DNA (B). Plasmid DNA

Agarose gel electrophoresis analysis of genomic and plasmid DNA: Plate

6.3(A) shows the gel pictures of isolated DNA; Lane 1, 2 & 3 are

samplesVREF1, VREF2 & VREF4 respectively. Plate 6.3 (B) shows the

gel picture of plasmids isolated from samples VREF 1 & VREF 4.

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Results of the phenotypic analysis for vancomycin resistance was,

confirmed by PCR analysis using primers specific to vancomycin resistance.

The isolate with vanA phenotype formed expected PCR product of 900bp

corresponding to the size of vanA gene. Similarly the PCR using primers

specific to vanB gene formed product of 700 bp size (Plates 6.4(A) & (B) .

Plate 6.4 .PCR detection of vanA and vanB genes of VREF

(A) (B)

Plate 6.4.(A): Agarose gel electrophoresis analysis & PCR amplification of

vanB genes from various strains of VREF are shown in plate 6.4.(A).Lane

1:Molecular size markers(100bp),Lane 2:VREF1,Lane 3:VREF 4.

Plate 6.4 (B): Agarose gel electrophoresis analysis & PCR amplification of

vanA gene from VREF2 is shown in Plate 6.4 (B). Lane 1: Molecular size

markers (100bp), Lane 2:VREF2.


To confirm the accuracy of PCR product formed, the product was gel

purified and subjected to DNA sequencing. This part of the work was

carried out at Sci Genome, Cochin. The sequence data of both vanA and

vanB genes were then subjected to BLAST analysis in which vanA gene

sequence showed its maximum identity to 922bp and that of vanB showed its

maximum identity to 706bp.

To confirm the plasmid borne nature and transferability of vanB

gene, the isolated plasmid was also used for mating experiments which

resulted in the formation of vancomycin resistant transconjugants. This was

confirmed by the growth of tranconjugants in BEA agar- media containing

16 µg/mL concentrations of vancomycin and 20 µg/mL concentrations of

rifampicin. Transconjugants showed same MIC as that of the donor E.

faecium and the presence of plasmid in the transconjugants was also

confirmed by plasmids isolation. The efficiency of conjugation ranged from

10-6 to 10-7 per donor cell.

Plate.4. illustrates the gel pictures of RAPD analysis of VRREF

samples.

146 Chapter 6

Plate 6.5: RAPD-PCR profile of VREF 6.5(A) 6.5 (B)

6.5(C) 6.5(D)

Plate 6.5. RAPD-PCR profiles among VREF isolates of clinical and chicken sources generated with each oligonucleotide. OPK7, OPK11, OPA14 and OPAA 17 in plate 6.5(A) & 6.5(B). RAPD-PCR profiles among VREF isolates of clinical and chicken sources generated with each oligonucleotide OPE6, OPL7, OPK12, and OPBG19 in plate 6.5(C) & 6.5(D). (M: 100 bp DNA ladder. Lanes 2, 4, 6 & 8: VREF1isolates from clinical sources; lanes 3, 5, 7 & 9: VREF4 isolate from chicken sources.

The genetic diversity analysis shown by RAPD had difference in

bands when oligonucleotides OPK11, OPK12 and OPBG19 were used for

the PCR.


6.5. DISCUSSION

Analysis of antibiotic resistance pattern shown by the isolates was

included in this study. The results of this study indicate that multi-drug

resistance is common among isolates of enterococci from all sources.

Penicillin resistance was high, nevertheless it was less in isolates from

human faeces compared to the other sources (P<0.05). According to some

earlier studies, a high percentage of enterococci from poultry and water were

resistant to penicillin (Garcia-Migura et al., 2005; Grammenou et al., 2006).

The percentage incidence of penicillin resistance observed in clinical isolates

was similar with the earlier findings reported across India and in some other

countries (Mendiratta et al., 2008). In this study the oxacillin resistance was

very common. Glew et al., (1975) have reported that the penicillinase-

resistant penicillins were less active than penicillin against enterococci.

Though ampicillin is the drug of choice for treating enterococcal

infections, most studies conducted across India and other parts of the world

have reported high ampicillin resistance (Randhawa et al., 2004). However

in this study the incidence of ampicillin resistance among the clinical isolates

of E.faecalis and E.faecium were much lower than these studies. Until

recently, ARE were recovered sporadically from animals and humans outside

the nosocomial environment (Biavasco et al., 2007). But in this study

enterococci from water had a higher rate of ampicillin resistance. While

considering the MIC of ampicillin in the present study, it was in the range of

0.5 to32 µg/mL. But E. faecium strains expressing very high levels of

ampicillin resistance (MIC >128 µg/mL) have been reported by Grayson et

al., (1991). Nevertheless in this study no such high level resistance was

observed.

148 Chapter 6

Though a high percentage of the isolates showed antibiotic resistance

to penicillin, none expressed detectable beta-lactamase activity when tested

with cefinase disks. It is quite possible that the penicillin resistance observed

could be due to the altered penicillin binding protein. Many studies

conducted in northern part of India have reported beta lactamase activity

(Mohanty et al., 2005).

In the present study a high percentage of E.faecium and E.faecalis

isolates from clinical sources were resistant to low and high levels of

gentamicin. Earlier studies showed both low and high level of gentamicin

resistance among clinical isolates of Enterococi (Udo et al., 2003).

Aminoglycosides are used in clinical practice because of its synergistic effect

with cell wall synthesis inhibitors like penicillin or vancomycin. Rosvoll,

(2012) also found the high incidence of HLGR among E.faecium isolates

from clinical sources. Hence the aminoglycoside resistance is of great

concern in the treatment. High-level aminoglycoside resistance may be

acquired by chromosomal mutation or by acquisition of plasmids encoding

aminoglycoside-modifying enzymes (Krogstad et al., 1978; Eliopoulos et al.,

1984). Therefore, HLGR deserve further attention.

In this study only a few enterococcal isolates from chicken sources

exhibited resistance to gentamicin. The degree of high level resistance was

nil in these isolates. Similar results were seen in another study (Hayes et al.,

2004). Gentamicin is seldom used in veterinary medicine and the low

incidence of gentamicin resistance observed in enterococci from chicken

sources can due to this.

In this study the enterococcal strains from all sources except human

faecal isolates showed resistance to amoxyclav. Amoxyclav resistance was


distributed in 14% of E. faecalis and 27% of E. faecium of clinical origin.

These observations strengthen the reports of other studies from India

(Miskeen and Deodhar, 2002), in which amoxyclav resistance was found to

be present, though studies from West Indies and Iran have reported 100%

sensitivity for amoxyclav (Orrett and Connors, 2000).

In this study a high percentage of enterococci from chickens were

resistant to tetracycline, compared to clinical sources. There are similar

reports from U.S. and abroad (Wiggins, 1996); (Yoshimura et al., 2000).

Their resistance level has been attributed to the extensive use of broad-

spectrum antibiotics for disease prevention in poultry production.

Chloramphenicol resistance in this study was found to be

comparatively more in environmental isolates of E.faecalis than from other

sources. This can be correlated with the less frequent use of chloramphenicol

for human treatment, because of its side effects. Despite of this low use in

humans, chloramphenicol resistance occurred in human enterococci isolates

of this and other studies in Denmark (Aarestrup et al., 2000). The use of

other antibiotics (e.g., penicillins, macrolides, and cephalosporins) appears to

drive chloramphenicol resistance, which is often a part of gene clusters that

encode for multidrug resistance. Chloramphenicol-resistant strains spread to

people from cattle through food have been discussed by Davis et al., (1999).

Clindamycin resistance in all the isolates was very high in this study.

Similar high resistance against clindamycin has been reported in some other

studies (Singh et al., 2002). Graham et al., (2009) also had found that all

enterococcal isolates had resistance to clindamycin.

Erythromycin resistance was more in enterococcal isolates from

chicken than in clinical isolates. The rapid development of erythromycin

150 Chapter 6

resistance in the poultry production environment as the result of medicated

feed use has been documented (Aarestrup et al., 2000). Resistance displayed

by clinical isolates may be due to the over use of erythromycin and other

macrolides.

A high incidence of ciprofloxacin resistance was shown by the water

and clinical isolates. The prevalence of resistance to fluoroquinolones in

human infections acquired from animals through the food chain is increasing.

Resistance to ciprofloxacin has emerged in enterococci over the last few

years and is now spreading clonally (Schaberg et al., 1992). High-level

resistance to fluoroquinolones is due to mutations in regions encoding

subunits of DNA gyrase and topoisomerase IV.

Nitrofurantoin is a successful antibiotic used to treat urinary tract

infections. Resistance to nitrofurantoin was observed to be more in clinical

isolates compared to other sources. Among the enterococcal isolates tested in

this study 7% of E.faecalis and 19% of E.faecium were resistant to

nitrofurantoin. Nitrofurantoin resistance was very less in nonclinical isolates.

Resistance was found only in low percentages or is absent (Rudy et al.,

2004).

None of the isolates in the present study was resistant to linezolid.

However there were few reports of linezolid resistance from other parts of

world. Many incidences of linezolid-resistant enterococci (LRE) have been

reported (Seedat et al., 2006; Auckland et al., 2002). There are even reports

of linezolid-resistant and vancomycin-resistant E. faecium (LRVRE)

(Gonzales et al., 2001).

Resistance towards glycopeptides, like vancomycin and teicoplanin

was significantly low and was shown only by the isolates of E.faecium.


Those phenotypic expressions of vanA gene is high level resistance to

vancomycin and resistance to teicoplanin and that of vanB gene is with low

levels of resistance to these antibiotics. Only 6 isolates of VRE (3vanA 3

vanB phenotypes) could be isolated from clinical sources in this study.

Although in many studies conducted in other countries high percentage of

glycopeptides resistance was reported (Pfaller et al., 1998 & Perlada et al.,

1997), the result of the present study is in accordance with the situation in

northern India where the incidence was low (Mathur et al., 2003& Ghoshal

et al., 2006). Vancomycin resistant enterococci have the ability to spread and

cause hospital outbreaks (Patel, 2003).

In this study from chicken sources, 20 vancomycin resistant

E.faecium isolates were obtained and all exhibited vanB type of vancomycin

resistance. In discrepancy, vanA was the most frequently recovered

vancomycin resistant phenotype observed in isolates from animal sources in

earlier studies (Bates et al., 1994). The low prevalence of vancomycin-

resistant E. faecium in normal intestinal flora of humans is probably due to

low glycopeptide consumption in humans.

Genotypic analysis of vancomycin resistance has shown that the same

genotype was observed in accordance with the resistant phenotype. Results

of PCR amplification on 3 strains verified that 1 clinical strain had vanA & 1

had vanB gene and the chicken isolate also had vanB gene. The occurrence

of a phenotype not in constant with the genotype has been reported (Bell et

al., 1998). In this study the phenotypic characters and genotypes were

conforming in the two selected isolates. Also the results of the study confirm

vanB genes as plasmid coded and vanA as chromosomally encoded. A

previous report also indicates that the vanB resistance determinants may be

plasmid carried (Showsh, 2001).

152 Chapter 6

The results of the study also confirmed the ability of vanB coding

plasmids of chicken VREF (sample no.210) to transfer conjugatively to other

enterococci by broth mating. The recovery of transconjugants with the same

MIC as that of the donor E. faecium proved this. Similar to the current

study, vanB transfer has also been noticed in vivo and in vitro in previous

reports (Dahl et al., 2007). The results of some other studies are also in

agreement with this study where transferable vanB plasmids from strains of

E. faecium, transferred at rates of 10−7 to 2 × 10−7/ recipient (Rice et al.,

1998). Also there are studies demonstrating gene transfer by conjugation

from E.faecium to E. faecalis (Leclercq et al., 1989). In addition to this,

Noble et al., (1992) reported transfer of genes from E. faecalis to S. aureus.

In this study, VREF strains harboring vanB genes from chicken and

clinical sources were found to be polymorphic by RAPD typing. Earlier

studies proved that the RAPD methods as a powerful tool to investigate VRE

isolates in cases of nosocomial infection (Barbier et al.,1996) Results of

recent studies also support RAPD as effective tool for epidemiological

investigations of enterococci (Werner et al., 2003). The RAPD analysis

clearly indicated that the strains from chicken samples and clinical sources

were not identical.

The answer to the question on the origin of human VRE remains

obscure because of the lack of evidence for the spread of strains from

animals to humans (Donnelly et al., 1996). However there are studies also

explaining with strong evidence to support infection potential of animal

enterococci (Iversen et al., 2002). Even though the present study excludes

the possibility of such a spread from chicken sources, there is the possibility

of horizontal transfer of vanB vancomycin resistance plasmids by


conjugation to other enterococcal strains and this was in agreement with the

findings of previous studies (Chow et al., 1993; Carias et al., 1998,).

The low prevalence of vancomycin resistance among the isolates in

this study indicates that vancomycin is still retaining its therapeutic efficacy

against the majority of enterococci. Since linezolid resistance is absent in

these isolates, it can also be used in treatment.

This work also establishes a baseline of resistance among

Enterococcus species in this part of the country which will be useful in

monitoring the dynamics of resistance. Drug resistant enterococci from

different sources can not only cause infections in immune compromised

hosts, but also may serve as important reservoirs of resistance genes. The

present study points to the necessity to control the spread of the antibiotic

resistance, by judicious use of antibiotics. Use of antibiotics in veterinary

therapy and bacterial infection prevention in animals should be minimized by

improving methods of animal husbandry and disease eradication.

antibiotic resistance of the enterococcal isolates...

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