This article was downloaded by: [Samiran Bandyopadhyay]On: 04 December 2014, At: 20:16Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: MortimerHouse, 37-41 Mortimer Street, London W1T 3JH, UK

Veterinary QuarterlyPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/tveq20

Co-infection of methicillin-resistant Staphylococcusepidermidis, methicillin-resistant Staphylococcusaureus and extended spectrum β-lactamaseproducing Escherichia coli in bovine mastitis – threecases reported from IndiaSamiran Bandyopadhyaya, Indranil Samantab, Debaraj Bhattacharyyaa, Pramod KumarNandaa, Debasish Kara, Jayanta Chowdhuryc, Premanshu Dandapata, Arun Kumar Dasa,Nayan Batuld, Bimalendu Mondala, Tapan Kumar Duttae, Gunjan Dase, Bikash ChandraDasa, Syamal Naskara, Uttam Kumar Bandyopadhyaya, Suresh Chandra Dasa & SubhasishBandyopadhyaya

a Indian Veterinary Research Institute, Eastern Regional Station, 37 Belgachia Road,Kolkata - 700 037, Indiab Department of Veterinary Microbiology, West Bengal University of Animal and FisherySciences, Kolkata, Indiac State Livestock Farm, Kalyani, Indiad Department of Veterinary Medicine, West Bengal University of Animal and FisherySciences, Kolkata, Indiae College of Veterinary Sciences, Central Agricultural University, Aizwal, IndiaPublished online: 02 Dec 2014.

To cite this article: Samiran Bandyopadhyay, Indranil Samanta, Debaraj Bhattacharyya, Pramod Kumar Nanda,Debasish Kar, Jayanta Chowdhury, Premanshu Dandapat, Arun Kumar Das, Nayan Batul, Bimalendu Mondal, TapanKumar Dutta, Gunjan Das, Bikash Chandra Das, Syamal Naskar, Uttam Kumar Bandyopadhyay, Suresh Chandra Das &Subhasish Bandyopadhyay (2014): Co-infection of methicillin-resistant Staphylococcus epidermidis, methicillin-resistantStaphylococcus aureus and extended spectrum β-lactamase producing Escherichia coli in bovine mastitis – three casesreported from India, Veterinary Quarterly, DOI: 10.1080/01652176.2014.984365

To link to this article: http://dx.doi.org/10.1080/01652176.2014.984365

PLEASE SCROLL DOWN FOR ARTICLE

Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) containedin the publications on our platform. However, Taylor & Francis, our agents, and our licensors make norepresentations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose ofthe Content. Any opinions and views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be reliedupon and should be independently verified with primary sources of information. Taylor and Francis shallnot be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and otherliabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to orarising out of the use of the Content.

This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in anyform to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions

CASE SERIES

Co-infection of methicillin-resistant Staphylococcus epidermidis, methicillin-resistant

Staphylococcus aureus and extended spectrum b-lactamase producing Escherichia coli

in bovine mastitis � three cases reported from India

Samiran Bandyopadhyaya*, Indranil Samantab, Debaraj Bhattacharyyaa, Pramod Kumar Nandaa, Debasish Kara, Jayanta

Chowdhuryc, Premanshu Dandapata, Arun Kumar Dasa, Nayan Batuld, Bimalendu Mondala, Tapan Kumar Duttae,

Gunjan Dase, Bikash Chandra Dasa, Syamal Naskara, Uttam Kumar Bandyopadhyaya, Suresh Chandra Dasa

and Subhasish Bandyopadhyaya

aIndian Veterinary Research Institute, Eastern Regional Station, 37 Belgachia Road, Kolkata - 700 037, India; bDepartment ofVeterinary Microbiology, West Bengal University of Animal and Fishery Sciences, Kolkata, India; cState Livestock Farm, Kalyani,India; dDepartment of Veterinary Medicine, West Bengal University of Animal and Fishery Sciences, Kolkata, India; eCollege of

Veterinary Sciences, Central Agricultural University, Aizwal, India

(Received 17 April 2014; accepted 2 November 2014)

Emergence of antimicrobial resistance among bovine mastitis pathogens is the major cause of frequent therapeutic failureand a cause of concern for veterinary practitioners. This study describes intra-mammary infection of methicillin-resistantStaphylococcus epidermidis (MRSE), methicillin-resistant Staphylococcus aureus (MRSA) and extended spectrumb-lactamase (ESBL) producing Escherichia coli in two Holstein Friesian crossbred cows with subclinical mastitis and onenon-descript cow with clinical mastitis in two different districts of West Bengal, India. In total, three MRSE, one MRSAand three ESBL producing E. coli were isolated from these cases. Both the crossbreds were detected with MRSE (HFSE1and HFSE2) and ESBL producing E. coli (HFEC1 and HFEC2), whereas, simultaneous infection of three pathogens viz.MRSA (NDSA1), MRSE (NDSE1) and ESBL producing E. coli (NDEC1) was found in the non-descript cow. Themethicillin-resistant isolates possessed mecA gene and exhibited resistance to various antibiotics such as amikacin,tetracycline and glycopeptides. The ESBL producers were positive for blaCTX-M and blaTEM genes; in addition, HFEC1and HFEC2 were positive for blaSHV and possessed the genes for class I integron (int1), sulphonamide resistance (sul1),quinolone resistance (qnrS) and other virulence factors (papC, iucD and ESTA1). All the ESBL producers exhibitedresistance to a variety of antibiotics tested including third- and fourth-generation cephalosporins and were alsointermediately resistant to carbapenems. This is the first ever report on simultaneous occurrence of MRSE, MRSA andESBL producing E. coli in bovine mastitis indicating a major concern for dairy industry and public health as well.

Keywords: bovine; cow; India; ESBL; mastitis; MRSA; MRSE

Introduction

Two Holstein Friesian (HF) crossbred cows (HFC1 and

HFC2) and one non-descript cow (NDC, without any

progeny record) from Nadia and Kolkata districts of West

Bengal, India, respectively, were reported with abnormal

udder secretion and drop in milk yield (Table 1). In all

three cows (HFC1, HFC2 and NDC), appearance of flakes

was noticed in milk. Besides, NDC was detected off-fed

for three days with mild rise of temperature (39.4 �C), hotpainful mildly swollen udder besides appearance of blood

clots in milk (Figure 1).

Milk samples, collected from all the animals, were

subjected to White Slide Test (WST), California Mastitis

Test (CMT) and Somatic Cell Count (SCC) following

standard procedure (Radostits et al. 2000). Diluted milk

(10¡2 to 10¡3 dilution) was incubated at 37 �C for 24 h

for determination of total viable bacterial count (TVBC)

and expressed in cfu/ml. The blood collected via jugular

vein and the smear prepared from ear vein puncture were

checked for routine haematological examination and

blood protozoa, respectively.

For direct examination of etiological bacteria, the smear

prepared from the individual milk samples was examined

microscopically following Gram’s staining. In the stained

smear, both Gram-positive cocci in bunches and Gram-neg-

ative bacilli were detected in all samples. For isolation of

the bacteria, milk samples were inoculated separately into

the nutrient broth (HiMedia, Mumbai, India) and incubated

overnight at 37 �C. The growth in nutrient broth was fur-

ther transferred into Baird Parker Agar (HiMedia, Mumbai,

India) with egg yolk emulsion. All the distinct black-col-

oured colonies with or without opalescence were further

subjected to standard biochemical tests, such as catalase,

coagulase and oxidase tests, glucose and mannitol fermen-

tation and lecithinase reactions (Quinn et al. 1994). For iso-

lation of Gram-negative bacilli, the growths in nutrient

broth were inoculated into Mac-Conkey agar (HiMedia,

Mumbai, India) and further into Eosin Methylene Blue

agar (HiMedia, Mumbai, India). The colonies with charac-

teristic metallic sheen were further subjected to standard

biochemical tests, such as catalase, oxidase, indole, methyl-

red, VP, citrate and urease (Quinn et al. 1994).

*Corresponding author. Email: samiranvet@gmail.com, sbandyo@ivri.res.in

� 2014 Taylor & Francis

Veterinary Quarterly, 2014

http://dx.doi.org/10.1080/01652176.2014.984365

Dow

nloa

ded

by [

Sam

iran

Ban

dyop

adhy

ay]

at 2

0:16

04

Dec

embe

r 20

14

All the biochemically confirmed Staphylococcus iso-

lates (11) were examined for their sensitivity to methicil-

lin (Met, 5 mg), oxacillin (Ox, 1 mg), cefoxitin (Cx,

30 mg), ciprofloxacin (Cf, 30 mg), amoxicillin and clavu-

lanic acid (AMC, 30 mg), amikacin (AK, 30 mg), tetracy-

cline (TE, 30 mg), vancomycin (VA, 30 mg), teicoplanin

(TEI, 30 mg) and tigecycline (TGC, 15 mg) by standard

disc diffusion assay (CLSI 2014). Minimum inhibitory

concentration (MIC) for the drugs oxacillin, cefoxitin and

vancomycin was measured using Ezy-MIC-strips (HiMedia,

Mumbai, India) according to the manufacturer’s instruc-

tions. Methicillin resistance was also confirmed by the

growth of the Staphylococcus isolates in oxacillin and

cefoxitin supplemented media (Hichrome MeRSA Agar,

HiMedia, Mumbai, India). For determination of vancomy-

cin resistance, isolates with zone diameters � 15 mm in

disc diffusion assay and for which vancomycin MICs

were �16 mg/mL (for Staphylococcus aureus)/�32 mg/

mL (for S. epidermidis) were classified as vancomycin-

resistant, whereas the isolates with zone diameters �15 mm and for which vancomycin MICs were 4�8 mg/

mL (for S. aureus)/ 8�16 mg/mL (for S. epidermidis)

were classified as vancomycin-intermediate (http://www.

cdc.gov). For determination of tigecycline susceptibility,

interpretation of zone diameters of all the Staphylococcus

isolates was done using the US FDA (Food and Drug

Administration) tigecycline susceptible breakpoints listed

for S. aureus. Isolates with zone diameter of �19 mm

only were classified as susceptible (Behera et al. 2009).

The isolates exhibiting resistance to methicillin, cefoxitin

and oxacillin were further screened for the presence of

methicillin resistance (mecA), coagulase (coa) and ther-

monuclease (nuc) genes by PCR as described earlier

(Wong et al. 2010).

All the seven E. coli isolates recovered from three

cows were further assessed for their sensitivity/resistance

to ceftazidime (CAZ, 30 mg), cefotaxime (CTX, 30 mg),

ceftriaxone (CTR, 30 mg), cefepime (CPM, 30 mg),

amoxicillin and clavulanic acid (AMC, 30/10 mg), tetra-

cycline (TE, 30 mg), gentamicin (G, 10 mg), amikacin

(AK, 30 mg), co-trimoxazole (Co, 25 mg), ciprofloxacin

(Cf, 30 mg), chloramphenicol (C, 30 mg), imipenem

(IMP, 10 mg), ertapenem (ETP, 10 mg) and meropenem

Figure 1. Mildly swollen udder of non-descript cattle.

Table1.

Clinico-pathologicalprofileofthreecasesofbovinemastitis.

Laboratory

findings

Bacteriology

Patients

District

Disease

Clinicalfeatures

WST�

CMT�

SCC� /ml

(Log10)

Haematology

Totalviable

bacterialcount

(cfu/m

l)

Bacteriaisolated

(number

ofisolatesin

parenthesis)

Holstein

Friesian

cross

1(H

FC1)

Nadia

Subclinical

mastitis

Notsignificant,

occasionalflakes

inmilkfrom

all

thequarters

CC

5.93

Nosignificant

change

0.05£

106

Staphylococcus

epidermidis(3),

Escherichia

coli(2)

Holstein

Friesian

cross

2(H

FC2)

Nadia

Subclinical

mastitis

Notsignificant,

occasionalflakes

inmilkfrom

all

thequarters

CC

5.85

Nosignificant

change

0.05£

106

S.epidermidis(2),

E.coli(2)

Non-descript

cow

(NDC)

Kolkata

Clinical

mastitis

Hotpainfulmildly

swollen

udder,

fever,off-fed

for

threedays,blood

clotandflakes

inmilk

CCC

C6.28

Neutrophilia

(36%

DLC)

2.1£

106

S.aureus(3),

S.epidermidis(3),

E.coli(3)

� WST:WhiteSlideTest;CMT:CaliforniaMastitisTest;SCC:SomaticCellCount.

2 S. Bandyopadhyay et al.

Dow

nloa

ded

by [

Sam

iran

Ban

dyop

adhy

ay]

at 2

0:16

04

Dec

embe

r 20

14

(MRP, 10 mg). MIC values of all isolates were determined

for ceftazidime, cefepime, cefotaxime, and ceftriaxone

using Hi-comb MIC test (HiMedia, Mumbai, India). The

isolates with zone of inhibition beyond the Clinical and

Laboratory Standards Institute (CLSI) recommended cut-

off values or with a breakpoint of MIC > 2 mg/ml for the

third- or fourth-generation cephalosporins were further

subjected to ESBL phenotypic determination by com-

bined disc method and ESBL E-test (CLSI 2014). The

combined disc method was performed using cefotaxime

(30 mg), ceftazidime (30 mg) and cefepime (30 mg) disc

alone or with clavulinic acid (10 mg) and an increase of

5 mm or more in the zone of inhibition in the presence of

clavulinic acid was considered positive for ESBL produc-

tion. ESBL E-test was performed using triple ESBL Ezy-

MIC-strip (HiMedia, Mumbai, India) and eightfold

increase in MIC in presence of b-lactamase inhibitors was

confirmed for ESBL production. All the three confirmed

ESBL producing E. coli isolates were subjected to modi-

fied hodge test (MHT) [http://www.cdc.gov] and disc dif-

fusion test with carbapenemase inhibitor [ethylene

diamine tetra-acetic acid (EDTA) and phenylboronic acid

(PBA)] impregnated agar as described by Birgy et al.

(2012). Furthermore, these ESBL producers were

screened for major b-lactamase-gene families blaCTX-M,

blaSHV, blaTEM, blaNDM OXA1 and OXA2 (Bhattacharjee

et al. 2007; Sarma and Ahmed 2010; Manchanda et al.

2011), plasmid‑mediated quinolone resistance (PMQR)

determinants (qnrA, qnrB, qnrS and aac(6 0)-ib-cr (Cie-

sielczuk et al. 2013), class I integron (int1), sulphonamide

resistance (sul1) genes (Lindstedt et al. 2003), cardinal

virulence markers for Shiga toxin producing E. coli (stx1and stx2), necrotoxigenic E. coliand extra-intestinal patho-

genic E. coli (ExPEC) by PCR as describer earlier (Paton

and Paton 1998; Van Bost et al. 2003).

Of the three suspected cases investigated, the cross-

breds (HFC1 and HFC2) were confirmed for subclinical

and the NDC for clinical mastitis by the WST, CMT and

SCC tests (Table 1). The haematogical parameters did not

reveal any significant change except neutrophilia in the

NDC.

The TVBC in milk was higher in the NDC than in the

crossbreds. In total, eight S. epidermidis, three S. aureus

and seven E. coli were isolated from these three cases of

bovine mastitis (Table 1). From HFC1, three S. epidermi-

dis were isolated of which one was methicillin resistant

(HFSE1) as revealed by disc diffusion assay and their

growth in Hichrome MeRSA agar. Of the two S. epidermi-

dis isolates from HFC2, one was methicillin resistant

(HFSE2). From the NDC with clinical mastitis, three S.

epidermidis and three S. aureus were isolated. Of them,

each of S. aureus (NDSA1) and S. epidermidis (NDSE1)

were methicillin resistant (Table 2).

The MRSE isolates (HFSE1, HFSE2 and NDSE1)

recovered in this study were phenotypically resistant to

methicillin, oxacillin, amoxicillin and clavulanic acid,

amikacin, tetracycline, vancomycin and not susceptible to

tigecycline (Table 2). On the other hand, MRSA isolate

(NDSA1) from NDC was phenotypically resistant to

methicillin, oxacillin, cefoxitin, amikacin, tetracycline,

vancomycin but was susceptible to tigecycline. In PCR,

all the four methicillin-resistant isolates harboured the

mecA gene and the S. aureus isolate (NDSA1) also carried

nuc and coa genes (Table 2).

Of the seven E. coli isolated in this study, only three

isolates (HFEC1, HFEC2 and NDEC1) were phenotypi-

cally confirmed as ESBL producers both by E-test and

combined disc method. All these three ESBL producers

exhibited resistance to the third- and fourth-generation

cephalosporins like ceftazidime, cefotaxime, ceftriaxone,

cefepime, along with amoxicillin and clavulanic acid, tet-

racycline, gentamicin, amikacin, co-trimoxazole and

chloramphenicol (Table 2). Furthermore, the isolates were

intermediately resistant to carbapenems (imipenem, mero-

penem and etrapenem). However, the isolates were found

negative in MHT and inhibitor based (EDTA and PBA)

phenotypic tests for carbapenemase or metallo-beta-lacta-

mase production. In contrast, all the ESBL producers and

methicillin-resistant Staphylococcus spp. isolated in this

study were sensitive or intermediately resistant to cipro-

floxacin except the MRSE isolate (NDSE1) obtained from

NDC.

Again, all the three ESBL producers (HFEC1, HFEC2

and NDEC1) possessed two major genes of b-lactamase

family (blaCTX-M, blaTEM) and the b-lactamase gene

blaSHV was detected in two isolates from HFEC1 and

HFEC2. Besides, the latter also possessed the genes for

class I integron (int1) and sulphonamide resistance (sul1).

However, none of the ESBL producers possessed studied

PMQR genes except HFEC2, which harboured qnrS.

Among other studied virulence factors, the papC,iucD

and ESTA1were detected in HFEC1 and HFEC2. In con-

trast, none of the isolates possessed the genes for Shiga

toxins (stx1, stx2) and cytotoxic necrotizing factor (cnf1,

cnf2) or other resistant traits investigated (OXA, NDM

and aac(60)-ib-cr.).Bovine mastitis continues to be a major challenge the

dairy industry is facing despite implementation of several

control programmes. The condition is further complicated

with the colonization and infection of drug-resistant path-

ogen(s) increasing the cost of treatment of sick animals.

Detection of such resistant pathogens is a major public

health concern due to the possibility of infection via con-

taminated unpasteurized milk and milk products intended

for human consumption. Although individual cases of

methicillin-resistant Staphylococcus and extended spec-

trum b-lactamase (ESBL) producing Enterobacteriaceae

have been reported in bovine mastitis in recent years

(Geser et al. 2012; Dahmen et al. 2013; Gindonis et al.

2013), there is no report apparently available on mixed

infection of three different kind of pathogens in bovine

mastitis.

This report describes co-infection of either or both

methicillin-resistant S. epidermidis (MRSE), methicillin-

resistant S. aureus (MRSA), and ESBL producing E. coli

harbouring virulence genes such as papC, ESTA1, and

iucD in three such cases of bovine mastitis in two cross-

breds and one NDC from West Bengal, India. The find-

ings revealed more association of MRSE than MRSA

with the bovine mastitis. Reports from other countries like

Veterinary Quarterly 3

Dow

nloa

ded

by [

Sam

iran

Ban

dyop

adhy

ay]

at 2

0:16

04

Dec

embe

r 20

14

Table2.

CharacterizationofMRSA,MRSEandESBLproducingE.coliisolatesfrom

bovinemastitis.

1.Methicillin-resistantStaphylococcusepidermidisandmethicillin-resistantStaphylococcusaureus

Genes

Antibioticresistance

profile

Isolates

mecA

nuc

coa

Met

Ox

Cx

Cf

AMC

AK

TE

VAa

TEI

TGCb

HFSE1

C¡

¡R

RI

IR

RR

RR

NS

HFSE2

C¡

¡R

RR

IR

RR

II

NS

NDSA1

CC

CR

RR

SI

RR

RI

S

NDSE1

C¡

¡R

RI

RR

RR

RR

NS

2.Extended

spectrum

b-lactamaseproducingEscherichia

coli

Antibioticresistance

profile

Isolates

CAZ

CTX

CPM

CTR

AMC

TE

GAK

Co

Cf

CIM

PMRP

ETP

HFEC1

RI

RI

RR

RR

RI

RI

II

HFEC2

RR

RR

RR

RR

RS

RI

II

NDEC1

RR

RR

RR

RR

RS

RI

SS

Resistance

andvirulence

genotypes

Phenotypicconfirm

ation

ofESBLproduction

Isolates

bla

CTX-M

bla

TEM

bla

SHV

sul1

int1

OXA1/2

NDM

qnrA

qnrB

qnrS

aac(60 )-ib-cr

CNF-1/2

stx1/2

Others

DDDT

E-Test

HFEC1

CC

CC

C¡

¡¡

¡¡

¡¡

¡ESTA1

CC

HFEC2

CC

CC

C¡

¡¡

¡C

¡¡

¡iucD

,papC,ESTA1

CC

NDEC1

CC

¡¡

¡¡

¡¡

¡¡

¡¡

¡¡

CC

Note:R:Resistant;S:Susceptible;NS:Not-susceptible;I:Interm

ediate.

Ceftazidim

e(CAZ,30mg),cefotaxim

e(CTX,30mg),ceftriaxone(CTR,30mg),cefepim

e(CPM,30mg),ciprofloxacin

(Cf,30mg),co-trimoxazole(Co,25mg),chloramphenicol(C,30mg),am

oxicillinandclavulanicacid

(AMC,30/10mg),tetracycline(TE,30mg),gentamicin

(G,10mg),am

ikacin

(AK,30mg),im

ipenem

(IMP,10mg),ertapenem

(ETP,10mg)andmeropenem

(MRP,10mg).

4 S. Bandyopadhyay et al.

Dow

nloa

ded

by [

Sam

iran

Ban

dyop

adhy

ay]

at 2

0:16

04

Dec

embe

r 20

14

the Netherlands, Finland and USA also indicate that

MRSE is an emerging etiological factor to cause bovine

mastitis (Sawant et al. 2009; Sampimon et al. 2011; Gin-

donis et al. 2013). Even the presence of MRSE alone with-

out any MRSA was also recorded from bovine subclinical

and clinical mastitis cases (Kot et al. 2012).

The ESBL-producing E. coli possessing blaCTX-M var-

iants were reported previously from mastitic cattle in

France, Japan and United Kingdom (Geser et al. 2012;

Dahmen et al. 2013; Ohnishi et al. 2013). Again, drug-

resistant Enterobacteriaceae possessing b-lactamase

encoding genes, such as blaTEM, blaSHV, blaCTX-M, blaOXAand other drug resistant determinants like qnr and class I

integron, were reported from bovine mastitis in Egypt

(Ahmed and Shimamoto 2011). However, there is hardly

any report of simultaneous infection of MRSA, MRSE

and ESBL-producing E. coli in the same mastitic cow

available to compare with the present findings. Further-

more, all the three ESBL producers from crossbreds were

found to carry virulence markers for ExPEC like papC,

iucD and ESTA1 which is rarely reported among the

ESBL producers from bovine mastitis, although such viru-

lence factors were reported among non-ESBL E. coli iso-

lates from clinical bovine mastitis in recent past (Suojala

et al. 2011). Detection of virulence genes required for

establishment of extra-intestinal infection in mastitic E.

coli isolates further makes the condition worse because

they can transmit the genes in commensal human E. coli

and convert them into extra-intestinal pathogenic E. coli

such as uropathogenic E. coli (Samanta et al. 2014.).

One of the ESBL producers (HFEC2) despite carrying

the plasmid-mediated (fluoro)quinolone resistance gene

(qnrS), was still sensitive to ciprofloxacin. This could be

due to the fact that qnr proteins are considered to confer

only low level of quinolone resistance by protecting the

bacterial DNA gyrase from (fluoro)quinolones. However,

in few instances, these can also facilitate higher resistance

during presence of (fluoro)quinolones at therapeutic con-

centration (Robicsek et al. 2006).

Similar to this finding, MRSE strains isolated from

mastitis milk samples of cattle also exhibited resistance to

tetracycline and different macrolides as reported by Kot

et al. (2012). Likewise, S. aureus isolated from goat milk

was also resistant to (oxy)tetracycline (Chu et al. 2012). It

is worrying that MRSA and MRSE isolates recovered in

this study also exhibited resistance or reduced susceptibil-

ity to glycopeptides or glycylcycline antibiotics like van-

comycin, teicoplanin and tigecycline, although these

drugs are never recommended in veterinary practice in

India. The glycopeptide resistance among S. aureus iso-

lates of animal origin is rarely found even though it is

widely detected in human (Thati et al. 2011). This could

be a grave public health concern as glycopeptides or gly-

cylcycline antibiotics are the choice of treatment in

MRSA infection in human beings. The ESBL producing

E. coli isolated in this study exhibited resistance to antibi-

otics belonging to multiple antimicrobial classes. Previ-

ously, ESBL-producing E. coli isolated from mastitic

milk showed resistance against chloramphenicol, genta-

micin and tetracycline in Switzerland (Geser et al. 2012).

However, the phenotypic expression of drug resistance in

ESBL producers is highly variable depending upon the

type of b-lactamase enzyme present and other resistance

traits they harbour in the plasmid, although multi-drug

resistance is commonly found among ESBL-producing E.

coli (Rakotonirina et al. 2013). Despite their intermediate

resistance to carbapenems, none of the ESBL producers

showed any evidence of MBL or carbapenemase produc-

tion either by phenotypic screening test or for blaNDMgene in PCR. Such resistance may be either due to high

expression of ESBL or in combination with reduced

expression of porin channels as suggested previously

(Birgy et al. 2012).

In conclusion, present investigation is an unique report

of simultaneous occurrence of MRSE, MRSA and ESBL-

producing E. coli in bovine mastitis. However, in absence

of any previous report or precise information of earlier

disease conditions/treatment of these cases, it is very diffi-

cult to comment, or conclude with regard to frequency of

such cases. Even though it seems to be an isolated case of

its own kind, such type of bovine intra-mammary infec-

tion is a cause of concern for public health.

Acknowledgements

The authors are thankful to the Director, IVRI, Izatnagar (UP)for providing necessary research facilities to carry out the pres-ent work.

Funding

The financial support received from DBT, Government of Indiais acknowledged.

Disclosure statement

The authors have no conflict of interest.

References

Ahmed AM, Shimamoto T. 2011. Molecular characterization ofantimicrobial resistance in Gram-negative bacteria isolatedfrom bovine mastitis in Egypt. Microbiol Immunol.55:318�327.

Behera B, Das A, Mathur P, Kapil A, Gadepalli R, Dhawan B.2009. Tigecycline susceptibility report from an Indian ter-tiary care hospital. Indian J Med Res. 129:446�450.

Bhattacharjee A, Sen MR, Anupurba S, Prakash P, Nath G. 2007.Detection of OXA-2 group extended-spectrum-b-lactamase-producing clinical isolates of Escherichia coli from India. JAntimicrob Chemother. 60:703�704.

Birgy A, Bidet P, Genel N, Doit C, Decre D, Arlet G, Bingen E.2012. Phenotypic screening of carbapenemases and associ-ated beta-lactamases in carbapenem-resistant Enterobacter-iaceae. J Clin Microbiol. 50:1295�1302.

Chu C, Yu C, Lee Y, Su Y. 2012. Genetically divergent methi-cillin-resistant Staphylococcus aureus and sec-dependentmastitis of dairy goats in Taiwan. BMC Vet Res. 8:39.

Ciesielczuk H, Hornsey M, Choi V, Woodford N, Wareham D.2013. Development and evaluation of a multiplex PCR foreight plasmid-mediated quinolone-resistance determinants. JMed Microbiol. 62:1823�1827.

CLSI. 2014. Performance standards for antimicrobial suscepti-bility testing. In: Clinical and Laboratory Standard InstituteWayne, PA: CLSI document; p. M100�S124.

Veterinary Quarterly 5

Dow

nloa

ded

by [

Sam

iran

Ban

dyop

adhy

ay]

at 2

0:16

04

Dec

embe

r 20

14

Dahmen S, Metayer V, Gay E, Madec JY, Haenni M. 2013.Characterization of extended-spectrum beta-lactamase(ESBL)-carrying plasmids and clones of Enterobacteriaceaecausing cattle mastitis in France. Vet Microbiol.162:793�799.

Geser N, Stephan R, H€achler H. 2012. Occurrence and character-istics of extended-spectrum b-lactamase (ESBL) producingEnterobacteriaceae in food producing animals, minced meatand raw milk. BMC Vet Res. 8:21.

Gindonis V, Taponen S, Myllyniemi AL, Pyorala S, NykasenojaS, Salmenlinna S, Lindholm L, Rantala M. 2013. Occurrenceand characterization of methicillin-resistant Staphylococcifrom bovine mastitis milk samples in Finland. Acta Veteri-naria Scandinavica. 55:61.

Kot B, Piechota M, Wolska K, Frankowska A, Zdunek E, BinekT, K»opotowska E, Antosiewicz M. 2012. Phenotypic andgenotypic antimicrobial resistance of Staphylococci frombovine milk. Polish J Vet Sci. 15:677�683.

Lindstedt B-A, Heir E, Nyga�rd I, Kapperud G. 2003. Characteri-

zation of class I integrons in clinical strains of Salmonellaenterica subsp. enterica serovars Typhimurium and Enteriti-dis from Norwegian hospitals. J Med Microbiol.52:141�149.

Manchanda V, Rai S, Gupta S, Rautela RS, Chopra R, RawatDS, Verma N, Singh NP, Kaur IR, Bhalla P. 2011. Develop-ment of TaqMan real-time polymerase chain reaction for thedetection of the newly emerging form of carbapenem resis-tance gene in clinical isolates of Escherichia coli, Klebsiellapneumoniae, and Acinetobacter baumannii. Indian J MedMicrobiol. 29:249�253.

Ohnishi M, Okatani AT, Harada K, Sawada T, Marumo K, Mur-akami M, Sato R, Esaki H, Shimura K, Kato H, et al. 2013.Genetic characteristics of CTX-M-type extended-spectrum-beta-lactamase (ESBL)-producing Enterobacteriaceaeinvolved in mastitis cases on Japanese dairy farms, 2007 to2011. J Clin Microbiol. 51:3117�3122.

Paton AW, Paton JC. 1998. Detection and characterization ofShiga toxigenic Escherichia coli by using multiplex PCRassays for stx 1, stx 2, eaeA, enterohemorrhagic E. coli hlyA,rfb O111, and rfb O157. J Clin Microbiol. 36:598�602.

Quinn PJ, Carter M, Markey B, Carter GR. 1994. Clinical Veter-inary Microbiology. St. Louis: Mosby-Wolfe.

Radostits O, Gay C, Blood D, Hinchcliff K. 2000. Veterinarymedicine: a textbook of the diseases of cattle, sheep, pigs,goats and horses. New York, NY: WB Saunders.

Rakotonirina HC, Garin B, Randrianirina F, Richard V, Talar-min A, Arlet G. 2013. Molecular characterization of multi-drug-resistant extended-spectrum beta-lactamase-producingEnterobacteriaceae isolated in Antananarivo, Madagascar.BMC Microbiol. 13:85.

Robicsek A, Jacoby GA, Hooper DC. 2006. The worldwideemergence of plasmid-mediated quinolone resistance. Lan-cet Infect Dis. 6:629�640.

Samanta I, Joardar SN, Das PK, Das P, Sar TK, Dutta TK, Ban-dyopadhyay S, Batabyal S, Isore DP. 2014. Virulence reper-toire, characterization, and antibiotic resistance patternanalysis of Escherichia coli isolated from backyard layersand their environment in India. Avian Dis. 58:39�45.

Sampimon O, Lam T, Mevius D, Schukken Y, Zadoks R. 2011.Antimicrobial susceptibility of coagulase-negative staphylo-cocci isolated from bovine milk samples. Vet Microbiol.150:173�179.

Sarma JB, Ahmed GU. 2010. Prevalence and risk factors for col-onisation with extended spectrum beta-lactamase producingEnterobacteriacae vis-a-vis usage of antimicrobials. IndianJ Med Microbiol. 28:217�220.

Sawant A, Gillespie B, Oliver S. 2009. Antimicrobial suscepti-bility of coagulase-negative Staphylococcus species isolatedfrom bovine milk. Vet Microbiol. 134:73�81.

Suojala L, Pohjanvirta T, Simojoki H, Myllyniemi AL, Pitkala A,Pelkonen S, Pyorala S. 2011. Phylogeny, virulence factorsand antimicrobial susceptibility of Escherichia coli isolated inclinical bovine mastitis. Vet Microbiol. 147:383�388.

Thati V, Shivannavar CT, Gaddad SM. 2011. Vancomycin resis-tance among methicillin resistant Staphylococcus aureusisolates from intensive care units of tertiary care hospitals inHyderabad. Indian J Med Res. 134:704�708.

Van Bost S, Jacquemin E, Oswald E, Mainil J. 2003. MultiplexPCRs for identification of necrotoxigenic Escherichia coli. JClin Microbiol. 41:4480�4482.

Wong H, Louie L, Lo RY, Simor AE. 2010. Characterization ofStaphylococcus aureus isolates with a partial or completeabsence of staphylococcal cassette chromosome elements. JClin Microbiol. 48:3525�3531.

6 S. Bandyopadhyay et al.

Dow

nloa

ded

by [

Sam

iran

Ban

dyop

adhy

ay]

at 2

0:16

04

Dec

embe

r 20

14