detection and antibiotic sensitivity testing of …

www.wjpps.com Vol 6, Issue 7, 2017.

1817

Rajashree et al. World Journal of Pharmacy and Pharmaceutical Sciences

DETECTION AND ANTIBIOTIC SENSITIVITY TESTING OF

BRUCELLA SPECIES

Apexa Suryawanshi and Dr. Rajashree Gandge*

1Livestock Development Officer, Nashik, Maharashtra.

2Associate Professor, Department of Microbiology, Bombay Veterinary College,

Parel, Mumbai- 400 012.

ABSTRACT

Infection with Brucella spp. continues to pose a human health risk

globally despite strides in eradicating the disease from domestic

animals. The timely and accurate diagnosis and treatment of human

brucellosis had been challenge for clinicians because of its non-

specific clinical features. Surveillance of disease or identifying

etiology is a key element for management of prevention and control

program. Therefore, the present study was designed to detect human

brucellosis in occupationally exposed humans to infected animals or

their products and to study antibiogram of Brucella spp. for detection of emergence of

resistance and to find out the suitable antibiotic for treatment of brucellosis. A total of 205

sera and 100 blood samples from animal handlers, farmers, milkers and practicing

veterinarians from different parts of Konkan and Western Maharashtra region were

processed. Overall seroprevalence of human brucellosis was found to be 16.58%, in a total of

205 sera samples tested by RBPT and STAT. Seroprevalence of brucellosis was found higher

in humans from Western Maharashtra region (23.52%) as compared to Konkan region

(9.09%). Only 4 Brucella isolates (6.6%) could be recovered from total 60 (22 seropositive +

38 seronegative) human whole blood samples attempted for isolation of Brucella spp.

employing biphasic media. Out of 4 Brucella spp. isolates recovered, 3 were identified as

Brucella abortus and 1 could not be identified upto its species level. Antibiotic susceptibility

test didn’t show development of drug resistance amongst Brucella spp. and isolates were

found sensitive to tetracycline, doxycycline and cephotaxim. PCR assays was carried out

using six different primers for detection of Brucella isolates at their genus and species level.

The PCR using BCSP31 (223 bp and 428 bp), DNAJ/BM (503 bp) and DNAJ/AB (388 bp)

*Corresponding Author

Dr. Rajashree Gandge

Livestock Development

Officer, Nashik, Maharashtra.

Article Received on

21 May 2017,

Revised on 13 June 2017,

Accepted on 2 July 2017

DOI: 10.20959/wjpps20177-9621

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

S SJIF Impact Factor 6.647

Volume 6, Issue 7, 1817-1829 Research Article ISSN 2278 – 4357


1818


primers were proved useful in confirming Brucella organisms at genus level. Whereas,

IS711/AB PCR produced abortus specific (Biotype1, 2, 4) amplification (498 bp) confirming

the isolates as B. abortus. None of 4 isolates showed B. melitensis specific amplification in

IS711/BM PCR. Direct detection of brucellosis by DNAJ/BM PCR from 100 human blood

samples revealed amplification product of 503 bp in 48% humans. PCR assays were found to

be most sensitive followed by serological and cultural methods in detection of human

brucellosis. Although, previous reports show prevalence of Br. melitensis in human

brucellosis; in present study Br. abortus was found to be predominant species existed in

chronic or subclinical form of occupationally exposed human population.

KEYWORDS: Bacterial isolation, Brucella abortus, Brucella melitensis, Human brucellosis,

PCR, Serology.

INTRODUCTION

Infection with Brucella spp. continues to pose a human health risk globally despite strides in

eradicating the disease from domestic animals. Brucellosis is one of the important diseases of

animals causing high economic losses in developing countries[9]

and having zoonotic

importance. The global burden of human brucellosis remains enormous; it causes more than

500,000 infections per year worldwide.[29]

The interest in brucellosis has been increasing

because of the growing phenomena of international tourism and migration, in addition to the

potential use of Brucella as a biological weapon.[23]

Although brucellosis is an important

disease in India, as per literature search it seems that, not much detailed research on human

brucellosis is carried out and only few recent studies have addressed the prevalence and

importance of brucellosis as a human disease problem in India. The timely and accurate

diagnosis and treatment of human brucellosis had been challenge for clinicians because of its

non-specific clinical features, slow growth rate in blood cultures and the complexity of its

serodiagnosis. However, with recent development in prompt diagnosis and treatment with

appropriate antibiotics may greatly reduce the time a patient may be incapacitated. Moreover,

surveillance of disease or identifying etiology is a key element for management of prevention

and control program. Therefore, the present research is designed with reference to study

prevalence of human brucellosis as an important re-emerging disease in occupationally

exposed humans by different diagnostic techniques.


1819


MATERIALS AND METHODS

Reference bacterial strains and Human specimens

The reference bacterial strains Brucella abortus (544) and Brucella melitensis Rev 1 were

used in present investigation. A total of 305 samples (205 sera + 100 whole blood) collected

from 205 humans formed material for present study (Table 1). Human isolates of Brucella

spp. recovered from human (volunteers) blood samples were used for characterization in the

present work.

Table 1: Showing different human specimens used and investigation carried.

Sr. No. Specimen Total no. of

specimens collected Investigation

Number of

samples processed

1 Whole blood in

EDTA 100

Whole blood

PCR 100

Isolation/ Culture 60

2 Serum 205 RBPT 205

STAT 205

Total 305

Primers

PCR assays were carried out using six different primers (Table 2) for identifing Brucella

isolates at genus and species level and also for detecting Brucella in human blood samples.

BCSP31,[4]

IS711/AB and IS711/BM were obtained from M/s Bangalore Genei, Bangalore

(India). Whereas, BCSP31 (31F/31R), DNAJ/BM, DNAJ/AB were designed primers.[20]

Serological tests

Serum samples were processed for detection of Brucella abortus antibodies by Rose Bengal

Plate Test (RBPT) and Standard Tube Agglutination Test (STAT) using Brucella abortus

agglutinating antigen obtained from Division of Biological Products, Indian Veterinary

Research Institute (I.V.R.I.); Izatnagar, Uttar Pradesh.

Table 2: Oligonucleotide primers for Molecular Characterization of Brucella.

Sr.

No

Name of the

PCR Primer sequence Reference

1. BCSP31 PCR

B4 -5’ : TGG-CTC-GGT-TGC-CAA-

TAT-CAA-'3 Baily et al. (1992)

B5- 5’: CGC-GCT-TGC-CTT-TCA-GGT-

CTG-‘3

2. BCSP31 PCR

31F-5’:TGC-GCG-TAT-CGT-TCT-TGA-

AGC-CTA-3’ Mahajan (2010)

31R-5’:TAT-CGA-GCT-TGA-TGA-GCT-


1820


TGC-CCT-3’

3. DNAJ/AB PCR

F- 5’: GGA-TAT-TTT-CGG-CGA-GAT-

GA-3’ Mahajan (2010)

R- 5’: GTC-CTC-GAT-ACC-TGT-GGG-

AA-3’

4. DNAJ/BM PCR

F-5’:TAC-GAA-ACC-CTG-AAA-GAC-

CCG-CAA-3’ Mahajan (2010)

R-5’:ATA-TTG-ACC-GAA-AGC-GAA-

CGC-TCC-3’

5. IS711/AB PCR

F-5’:TGC-CGA-TCA-CTT-AAG-GGC-

CTT-CAT- 3’ Bricker and

Halling (1994) R-5’:GAC-GAA-CGG-AAT-TTT-TCC-

AAT-CCC- 3’

6. IS711/BM PCR

F-5’:TGC-CGA-TCA-CTT-AAG-GGC-

CTT-CAT- 3’ Bricker and

Halling (1994) R-5’: AAA-TCG-CGT-CCT-TGC-TGG-

TCT-GA- 3’

Isolation, identification and antibiotic susceptibility test of Brucella spp. isolates

Isolation of Brucella spp. was carried out from blood sample using biphasic media[30]

and

further identification was done using different tests.[28]

Antibiotic susceptibility test of

Brucella isolates was carried out by disc diffusion method on Brucella agar medium.[17]

Antibiotics discs of Tetracycline (30 mcg), Erythromycin (15 mcg), Doxycycline (30 mcg),

Ciprofloxacin (5 mcg), Streptomycin (10 mcg), Co-trimoxazole (23.75 mcg) and Cephotaxim

(30 mcg) from Hi media were used.

Molecular Detection of Brucella spp. Isolates

Six PCR assays were carried out using genus specific primers viz, BCSP31 (B4/B5) and

(31F/31R) and species specific primers viz, IS711/AB and IS711/BM, DNAJ/AB and

DNAJ/BM for identification of Brucella isolates. Direct detection of Brucella spp. in blood

samples carried out by PCR amplification of DNAJ/BM gene. The amplification products

generating from all the PCR assays were evaluated by agarose gel electrophoresis in 1.5 %

agarose gel stained with ethidium bromide.[7]

Molecular Detection of Brucellosis in human blood samples

A total of 100 whole blood samples (34 seropositive + 66 seronegative) were investigated for

molecular detection of Brucella spp. directly in the extracted DNA from blood samples using

DNAJ/BM PCR assay.


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RESULTS AND DISCUSSION

Seroprevalence of Brucellosis in Humans

A total of 205 human sera were examined for presence of Brucella antibodies in which,

overall 34 cases were revealed seropositive together by RBPT (Plate 1) and STAT showing

16.58% seroprevalence of Brucellosis in Western Maharashtra and Konkan region.

Seroprevalence of brucellosis was observed higher in Western Maharashtra region (23.52%)

as compared to Konkan region (9.09%) as shown in table no. 3. RBPT (12.68%) and STAT

(14.14%) showed almost equal efficacy in detecting Brucella antibodies. However, three sera

samples which were positive in STAT were found negative by RBPT.

Table 3: Results of detection of Brucella antibodies by RBPT and STAT.

Region No. of samples

tested

Number of positive

samples (%) by RBPT

Number of positive

samples (%) by STAT

Konkan 154 14 (9.09) 19 (12.33)

Western

Maharashtra

51 12 (23.52) 10 (19.60)

Total 205 26 (12.68) 29 (14.14)

The seroprevalence of a present investigation (16.58%) correlated with the 15.72%; 15.69%;

19.83% seroprevalence reported.[1,2,31]

Similar efficacy of RBPT and STAT in detecting

Brucella antibodies was reported by some authours;[1,14,33]

however being IgM isotype of

antibody predominantly involved in agglutination reactions at neutral pH[25]

the false positive

reactions in STAT may occur due to such cross-reacting antibodies.[26]

Conventional characterization of Brucella isolates

Cultural isolation of Brucella spp. employing biphasic media (Plate 2) was attempted from

total 60 (22 seropositive + 38 seronegative) whole blood samples of human. Out of 60

samples processed for isolation, only 4 Brucella isolates (6.6%) could be recovered, of which

3 were from serologically positive and one was from serologically negative brucella sample.

Out of 4 human Brucella isolates recovered during present investigation, 3 were identified as

Brucella abortus whereas one isolate could not be identified upto its species level (Table 4).

Biotyping of 4 Brucella abortus isolates was attempted. The combined results of

conventional method and IS711/ AB PCR assay were used for detecting biotypes; however

from the results (Table 5) the biotype of any of the isolate could not be traced.


1822


Table 4: Characterization of Brucella isolates.

Sr. No. Name of tests

Isolates Reference strains

B.

abort

us

HM

N-

001

B.

abort

us

HM

N-

002

B.

abort

us

HM

N-0

03

B.

spp.

HM

N-

004

B.

abort

us

stra

in 5

44

B.m

elit

ensi

s

Rev

-1

1. Indole test - - - - - -

2. Urease test + + + + - +

3. Nitrate reduction + + + + + +

4. Catalase + + + + + +

5. Oxidase + + + + + +

6. Acriflavine test All isolates formed

homogeneous suspension

Homogeneous suspension.

Homo generous.

Susp. suspension

7. Reaction with anti-

Brucella std. serum + + + + + -

+ : Positive, - : Negative.

Table 5: Biotyping of Brucella isolates.

Sr.

No.

Isolates

CO2

requirement

H2S

production

Growth on media

containing

IS711

(AB)

PCR

Biotype Thionin* Fuchsin*

1. B. abortus

HMN - 001 + + + + +

B. abortus

biotype:

unidentified

2. B. abortus

HMN - 002 + + + + +

B. abortus

biotype:

unidentified

3. B. abortus

HMN - 003 + + + + +

B. abortus

biotype:

unidentified

4. B. spp.

HMN - 004 + + + + -

Brucella

spp.

Isolation of Brucella spp. from clinical specimens is considered to be the gold standard since

it is a direct evidence of infection; further, several groups working in India have attempted

isolation of Brucella spp. from different clinical specimens with varying degrees of success.

The blood samples have proved to be the useful specimen for isolation of Brucella spp. in

human beings and several researchers have successfully attempted isolation of Brucella spp.

from human blood samples for detection of Brucellosis.[34,27]

The probable reason for low % of isolation could be due to presence of few Brucella

organisms per unit of blood reducing chances of recovery of bacteria from blood, since the


1823


blood samples were not specifically collected critically at height of temperature. Moreover, it

has been suggested that in chronically infected cases when antibody levels are vanishing or in

case of recent exposure to low dose of brucella where there is no sufficient period for

development of immune response.[10]

Therefore reports of lower percentages viz. 0.77%,

8.33% & 0% of isolation of Brucella spp. from blood samples have been published[16,24,31]

which correlated with present study.

Amongst the above Brucella species known to infect humans, Brucella melitensis is the most

commonly reported,[21]

however Brucella abortus is also known to infect humans.[35]

The

disease in human is characterized by an acute bacteraemic phase and irregular fever, followed

by chronic stage that may extend over many years and may involve many tissues.[21]

In the present investigation, Brucella abortus species was found to be predominant in human

brucellosis. From the history of persons, it is revealed that brucellosis in occupationally

exposed animals seems to be existed in chronic or subclinical form in population, wherein no

severe acute symptoms of brucellosis observed in positive persons. Although, recently Br.

abortus spp. is not reported commonly in humans, there are past reports available, indicating

zoonotic importance of Br. Abortus.[32,35]

In antibiotic susceptibility test, all 4 isolates found sensitive to tetracycline, doxycycline,

ciprofloxacin, streptomycin and cephotaxim, whereas 2 isolates showed intermediate

sensitivity to erythromycin and one to co-trimoxazole. (Plate 3).

Table 6: Antibiotic susceptibility test of Human isolates.

Sr.

No Brucella Isolates

Antibiotics used

Tet

racy

clin

e

(10 m

cg)

Doxycy

clin

e

(30 m

cg)

Ery

thro

myci

n

(15 m

cg)

Cip

rofl

ox

aci

n

(30 m

cg)

Str

epto

my

cin

(10 m

cg)

Co-t

rim

oxa

zole

(10 m

cg)

Cep

hota

xim

(30 m

cg)

1. B. abortus HMN- 001 S S S S S S S

2. B. abortus HMN- 002 S S S S S I S

3. B. abortus HMN- 003 S S I S S I S

4. B. spp. HMN- 004 S S I S S I S

S = Sensitive, I = Intermediate

There is not yet resistance problem for classically recommended antibiotics targeted

to Brucella species reported, however antibiotic susceptibility patterns of Brucella spp.


1824


appear to vary geographically.[5]

Similar findings as in present study of not showing drug

resistance and effectiveness of above antibiotics in brucellosis was reported.[22,8]

The

emergence of drug resistance did not observe; may be due to lack of awareness for diagnosis

and treatment of brucellosis.

Molecular Detection of Brucella spp. isolates

All 4 human isolates and the reference strains B. abortus 544 and B. melitensis Rev 1 as

positive controls were included in PCR assay (Plate 4-8). Out of 4 isolates tested by genus

specific PCR assay BCSP31 published and newly designed primers, all isolates showed

specific amplification product of 223 bp and 428 bp respectively. Moreover, DNAJ/AB and

DNAJ/BM PCR also yielded 388 bp and 503 bp amplification products respectively in all

isolates including reference strains B.abortus 544 and B. meletensis Rev 1 confirming identity

of isolates as members of genus Brucella. In B. abortus specific IS711/AB PCR, out of 4

Brucella isolates, 3 showed an amplification product of 498 bp confirming their identity as B.

abortus. However, one isolate of Brucella did not yield abortus specific amplification

product. All isolates and reference strains were also simultaneously subjected to B. melitensis

specific IS711/BM PCR where in none of the isolate produced amplicon of 731 bp as

generated except reference strain B. melitensis Rev 1 suggesting that none of the isolates

belonged to Br. meletensis species.

A number of PCR assays targeting different regions of Brucella DNA viz. IS711[6]

heat shock

proteins like DnaJ[11]

etc. have been developed for identification of Brucella at genus and

species level. Sensitivity of PCR assay in detection of brucellosis has been reported by many

workers. The results of our study are in agreement with authors who employed IS711/AB and

IS711/BM PCR assays for identification of Brucella abortus and meletensis and found it to

be efficacious.[12,19,2]

The results of present investigations are concordant with the findings of

workers who used DNAJ PCR assays for detecting Brucella genus.[11,20]

Molecular Detection of Brucellosis from blood samples

A total of 100 whole blood samples (34 seropositive + 66 seronegative) were investigated for

molecular detection of Brucella spp. by DNAJ/BM PCR. An amplification product of 503 bp

was observed in 48 (48%) samples confirming the infection of human due to Brucella

species. Of 34 serologically positive samples, 32 (94.11%) yielded Brucella spp. specific

amplification product in PCR while two seropositive humans did not exhibit any

amplification. Out of 39 PCR positive blood samples, only 4 Brucella spp. (10.25%) isolates


1825


were recovered. PCR assay was found to be most sensitive followed by serological and

cultural methods in detection of human brucellosis during present investigation. Our results

correlated with the findings of investigators who suggested PCR to be of value in detection of

Brucella spp. from blood.[18]

As for other fastidious pathogens, amplification of DNA by PCR offers an alternative way of

diagnosis of brucellosis. Now a days genetic characterization using DNA based molecular

technique has been pursued for the rapid identification of Brucella. PCR has been applied to

detect Brucella DNA in varieties of clinical samples including tissues (mainly aborted

foetuses and associated maternal tissues), blood, milk and semen.[18,15,13]

The PCR based

methods are not only advantageous in being more sensitive than conventional methods in

diagnostic identification of the pathogen but can also facilitate characterization and typing of

the organism for studying epidemiology of infection.[3]

Besides being rapid and specific the

added advantage of these techniques is non-requirement of handling of virulent cultures

thereby reducing the safety concerns.

1 2 3 4 5 6 7

1000 bp

200 bp

100 bp

223 bp

1 2 3 4 5 6 7

Lane 1 : 100 bp ladder, 2: B. abortus 544, 3: HMN 001, 4: HMN 002

5: HMN 003 , 6:HMN 004 and 7: Negative control

300 bp

Plate 4: BCSP 31 (B4/B5) PCR. Plate 3: Antibiotic Sensitivity

Test of Brucella isolates

Plate 1: Detection of brucella

antibodies by Rose Bengal Plate Test.

Plate 2: Colonies of Brucella

spp. on biphasic media.


1826


1 2 3 4 5 6 7

Lane 1 : 100 bp ladder, 2: B. abortus 544, 3: HMN 001, 4:

HMN 002 5: HMN 003 , 6:HMN 004 and 7: Negative control

1000 bp

400 bp

100 bp

428 bp500 bp

1 2 3 4 5 6 7

1000 bp

300 bp

100 bp

400 bp 388 bp

Lane 1 : 100 bp ladder, 2: B. abortus 544, 3: HMN 001, 4:

HMN 002 5: HMN 003 , 6:HMN 004 and 7: Negative control

4321 5 6 7

1000 bp

100 bp

400 bp500 bp 503 bp

Lane 1 : 100 bp ladder, 2: B. abortus 544, 3: HMN 001, 4: HMN 002

5: HMN 003 , 6:HMN 004 and 7: Negative control

1000 bp

500 bp

100 bp

498 bp400 bp

1 2 3 4 5 6 7

Lane1:100 bp ladder, 2: B. abortus 544, lane 3 , 5 & 6: Br.

abortus positive samples.. Lane 7: negative control

1 2 3 4 5 6 7

CONCLUSIONS

Overall seroprevalence of Brucellosis was found to be 16.58%. Serological prevalence of

human brucellosis was found relatively higher in Western Maharashtra region (23.52%) as

compared to Konkan region (9.09%). Brucella abortus spp. was found to be predominant in

human brucellosis. Biotype 2 of Brucella abortus spp. was found prevalent in animal

brucellosis. In Antibiotic susceptibility test no marked difference in antibiogram pattern

amongst the isolates was found and none of the isolate showed resistance to any of antibiotics

tested in in vitro antibiotic susceptibility testing. PCR assays were found to be most sensitive

followed by serological and cultural methods in detection of human brucellosis.

Plate 8. IS711/AB PCR. Plate 7: DNAJ/BM PCR assay of

Brucella spp.

Plate 6: DNAJ/AB PCR

assay of Brucella spp.

Plate 5: BCSP 31 (31F/31R) PCR.


1827


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