PATHOLOGY IN PRACTICE NOVEMBER 2015 143

their way onto the plasmids of bacterialcells, allowing mutations and outbreaks to occur more frequently.

The majority of TEM and SHV mutantsare seen predominantly in Klebsiellapneumoniae, with Escherichia coli andother strains not being significantlyaffected; however, they are nowcommonly observed across variousspecies. Such enzymes are often referredto as classic β-lactamases as theygenerate narrow-spectrum β-lactamresistance against most penicillins, first-generation cephalosporins andcefoperozone. The CTX-M enzymes werefirst encountered in the early 2000s inKluyvera spp. but have spread via thetransfer of chromosomal genes onto the plasmids of various othermicroorganisms.2

Resistance achieved by horizontal genetransfer is far more concerning as it isable to disseminate across a population of bacterial cells more rapidly than simplemutation or vertical evolution. The locationof these enzymes allows them to be

transferred between surrounding bacteriaof the same or differing type and theproduction is constant, increasing thespread of resistance to different bacterialspecies and causing outbreak situations.Also propelled by horizontal gene transferis the rise and spread of multidrug-resistance (MDR); for example, ESBL-producing E. coli and Klebsiella spp. are increasingly being identified withadditional resistance enzymes that conferresistance to a multitude of antibioticclasses such as fluoroquinolones.

AmpC β-lactamasesAmpCs are primarily termedcephalosporinases as they hydrolysesecond- and third-generationcephalosporins and cephamycins, as wellas most β-lactams. They have alsoconferred resistance against somecarbapenems due to diminished porinexpression or porin loss.

AmpC enzymes may be chromosomal-intrinsic-mediated or plasmid-encoded,and those that are chromosomal may be inducible or derepressed. The ampRgene is responsible for the regulation ofchromosomal ampC expression byretaining it to a low-level. However, someampR genes are inducible in response toβ-lactams and other stimuli, leading tohyper-expression of the ampC gene andresulting in AmpC enzyme production.

Mutations may also occur in the ampRgene, resulting in derepressed mutants,and thus hyper-expression of the ampCgene. Complete derepression occurswhen the gene is not susceptible toinduction, but still omits high-levelexpression of ampC. Partial derepressionarises when ampC is expressed at greaterlevels than usual, but is still inducible.

Carbapenemases For patients with ESBL/AmpC-complicated-infections, carbapenemantibiotics are often the last line ofdefence. They are the only surviving β-lactams that remain to demonstrateactivity against such enzymes, but with

It has been estimated that antibiotics addan average of 20 years to all of our lives.1

In the 87 years since the discovery ofpenicillin, a huge variety of antibioticshave been created. This has madepossible an array of innovative medicaladvances, including organ transplantationand cancer therapy, and helps to savemillions of lives every day.

While the development of resistantstrains of microorganisms to antimicrobialagents is a natural occurrence, theprocess has no doubt been acceleratedby the misuse and overuse of antibiotics.Bacteria are now developing anabundance of mechanisms that allowthem to confer resistance, resulting in the development of resistant strains anduntreatable ‘superbugs’.

Particularly worrying is the prevalenceof resistance in Gram-negative pathogens,as isolates producing extended-spectrumβ-lactamase (ESBLs), AmpC andcarbapenemase enzymes are rapidlydisseminating. This has resulted in limitedtherapeutic treatment options, renderinginconsequential infections life-threatening.

Extended-spectrum β-lactamasesExtended-spectrum β-lactamases areenzymes produced by Gram-negativebacteria, commonly Enterobacteriaceae,which render various β-lactam antibioticsinactive against microorganisms. A number of different ESBL genotypeshave been identified, with the three most prevalent being those of the TEM,SHV and CTX-M variants. The TEM andSHV genotypes are naturally occurringchromosomal enzymes that have made

MICROBIOLOGY

Antimicrobial resistance is regarded as one of the most

pressing problems facing 21st-century healthcare, such

that it is the focus of The Longitude Prize 2014. Here,

Erin Vaughan provides an overview and update.

Antimicrobial resistance:where are we now?

‘The development ofresistant strains ofmicroorganisms toantimicrobial agents is anatural occurrence, butthe process has beenaccelerated by misuse andoveruse of the agents’

PATHOLOGY IN PRACTICE NOVEMBER 2015144

to India and the Middle East, there havebeen incidents reported in Europe, theUSA, Australia and Canada.3 The NDM-1enzyme is particularly worrying asantibiotic options continue to decline,leaving colistin, tigecycline andfosfomycin among the few that remaineffective.3

Non-MBL carbapenemases includeKPC and OXA types. These differ fromMBLs as they have serine present at theiractive site. The KPC enzymes belong tomolecular class A and are able tohydrolyse β-lactams, but they are partiallyinhibited by classic β-lactamase inhibitorssuch as clavulanic acid and boronic acid.An endemic unfolding from the Klebsiellapneumoniae carbapenemase (KPC) wasinitially seen in Italy and this is now themost widespread carbapenemase

observed in Enterobacteriaceae acrossEurope. It has been determined thataround 60% and 15% of K. pneumoniaein Greece and Italy, respectively, are nowresistant to carbapenems via KPCenzymes.2

The OXA enzymes belong to molecularclass D and there is currently no specificinhibitor that is effective against OXA-type carbapenemases, as the usualβ-lactamase inhibitors are yet todemonstrate any success. Sodiumchloride has showed slight activity in vitroand also avibactam, although the latteralso inhibited KPC enzymes. Particularlyproblematic are the OXA-48carbapenemases, which express high-level resistance to penicillins and low-level resistance to carbapenems andβ-lactamase inhibitors. However, they are not resistant to broad-spectrumcephalosporins with cefapirin andcefotaxime, for example, remainingeffective. Originally emerging in North Africa, the Middle East and India, OXA-48s are increasingly beingidentified in Europe. The emergence of OXA-48 carbapenemases inEnterobacteriaceae is of increasingconcern, as they are difficult to detect due to their resistance/susceptibilityprofile.

the development of carbapenemases on the rise their effectiveness iscompromised.2 Carbapenemases are β-lactamases that confer resistance, viahydrolysis, to a number of β-lactamsincluding penicillins, cephalosporins,monobactams and carbapenems.

Carbapenemase-producingEnterobacteriaceae (CPE) are extremelydifficult to treat due to their diversesusceptibility profiles. The propagation of carbapenemases became prevalent in Europe in the late 1990s across theMediterranean and was largely seen inPseudomonas aeruginosa.

Metallo-β-lactamase (MBL)carbapenemases fall under molecularclass B and include IMP, VIM and NDMtypes, all of which have zinc at their activesite. As well as hydrolysing carbapenems,they are also resistant to a number of β-lactamase inhibitors such as clavulanicacid. However, due to the presence ofzinc at the active site, they are effectivelyinhibited by metal ion chelators such asEDTA.

The first Verona integron-encoded MBL(VIM) was observed in Italy in 1997, andwent on to cause an epidemic in Greecein the early 2000s.2 Epidemics caused bythe New Delhi MBL (NDM-1) have alsobeen seen globally. Originally confined

MICROBIOLOGY

Fig 1. Flow chart for laboratory detection of CPE using mastdiscs resistance range.

‘The OXA enzymes belongto molecular class D andthere is currently nospecific inhibitor that iseffective against OXA-typecarbapenemases’

IsolatedEnterobacteriaceae No carbapenemase

No carbapenemase PresumptiveOXA-48

AmpC + porin loss

MBL KPC

With microcolonies

YES

YES

YES

YESNO

NO

NO

Zonegenerated

Is theorganismresistant toTEM30C?

Does zone sizecomparisonindicatepositive result onD70C?

Screen with CAT-IDTM (D71C)

Confirmatory test for acquired

carbapenemase

NB: Identification to genus/specieslevel is desirable for interpretation of

resistance patterns

Confirm MBL and KPC using D70C+

presumptive ID of OXA-48 using TEM30C

Perform further work (eg modified Hodge test or

molecular testing)

Equivocal

just $1.7 billion was awarded to AMR.6

In addition, during 2008–13, a similarsituation was encountered in the UK, with just £95 million being spent on AMRresearch from a total £14 billion researchfund for bacteriology.

Jim O’Neill, chair of the AMR reviewteam, has proposed a global innovationfund of $2 billion to boost drugs anddiagnostics research over the next fiveyears, which doesn’t seem adequatebased on previous figures.6 As it can takeover 20 years before pharmaceuticalcompanies begin to profit from such investments, Jim O’Neill5 hasrecommended that a $2–3 billionreimbursement fee should be paid tocompanies that succeed in developingnovel antibiotics. It is believed this willdiscourage such companies fromaggressively driving sales uponcommercialisation, selling lower volumesof the antibiotic and thus delayingresistance.6

Utilising its 60 years’ experience ofdevelopment and manufacture ofantimicrobial sensitivity testing (AST)products, Mast now offers mastpharma, a range of services tailored to thepharmaceutical industry for the evaluationof novel antimicrobial compounds. Clinicaltrials of antimicrobials require the designof diagnostic devices to determinemicroorganism susceptibility before the

PATHOLOGY IN PRACTICE NOVEMBER 2015 145

Impact of antimicrobialresistance on healthcareAntimicrobial resistance (AMR) is anincreasing burden as it continues to havea significant impact on human health andthe economy. Around two million peoplebecome infected with resistant bacteria inthe USA alone every year, complicatingotherwise fairly simple infections,resulting in delayed recovery time,lengthier (expensive) hospital stays and an increased risk of mortality.4

Although AMR has long beenrecognised as a significant threat tohealthcare, and despite being responsiblefor at least 50,000 deaths per yearthroughout the USA and Europe,5 theissue did not gain internationalimportance until it became the focus forThe Longitude Prize 2014. This £10 millionprize fund, provided by Nesta, aims toaddress one of the greatest issues of ourtime. Nesta has now released a brief forthe invention of a cost-effective, accurate,rapid and easy-to-use diagnostic test forbacterial infections that will allow healthprofessionals worldwide to administer the right antibiotics at the right time.1

Additionally, the UK government hasalso played a key role in raising awareness of AMR, particularly since theappointment of Professor Dame SallyDavies as Chief Medical Officer in 2010.She has promoted antibiotic advocacy andfought to change public perception.Furthermore, the announcement of theAMR review by David Cameron in July2014 has propelled the issue into themedia spotlight. It remains unclear,however, whether this has had an impactor if AMR remains in the shadows cast by cancer, the human immunodeficiencyvirus (HIV) and other global threats tohealth and wealth.

Antimicrobial resistance researchOver the past decade, antibioticconsumption has increased by almost40%,5 but antibiotic development isalmost at a standstill. Pharmaceuticalcompanies are reluctant to invest in thedevelopment of new antibiotics, as thepotential return on investment is trivial in comparison to other highly profitabledrug products. We are at risk of entering a post-antibiotic era where commoninfections will become potentially fatal,routine operations will no longer bepossible and healthcare systems willrapidly decline.

During 2010–14, a massive $142.5billion was devoted to medical researchfrom the US National Institutes of Health.It is estimated that $14.5 billion and $26.5 billion were dedicated to HIV/AIDSand cancer research, respectively, while

MICROBIOLOGY

compound enters phase 2 and 3 trials.Accordingly, AST discs are proven to bethe most effective and validated methodto determine bacterial susceptibility inboth clinical and veterinary practice, andmust be commercially available at thelaunch of the compound to assure fullmarket penetration.

In the disc development phase(mastpharma Development) an AST disc containing a novel compound isformulated. Additionally, methods for theassay of its content are verified. Initialstress studies identify the most suitablecandidate formulation for the productionof stable, investigational use only ASTdiscs, meeting defined quality controlacceptance parameters. A minimum ofthree replicate batches of product aremanufactured and entered into real-timestability studies (mastpharma Stability).These studies are carried out undernormal storage and additional stressconditions which ensures the finalproduct retains effective performance forthe duration of its allocated shelf-life.

Antimicrobial resistance diagnosticsIt has been estimated that AMR couldcause 10 million deaths per year by 2050,equating to cumulative costs of $100trillion, if ignored, according to the Review on AMR team.5 It is imperative,now more than ever; that scientists areable to make a differential diagnosis withregard to which resistant mechanism ispresent to aid the antibiotic selectionpressure that laboratories are faced with daily. This will also help to conserveand reserve specific antibiotics (egcarbapenems) by providing tailoredtreatment to patients and ultimatelyreducing the risk of the dissemination of carbapenemases.

Molecular-based assays for thedetection of AMR have led to a number of important discoveries, including theidentification of the methicillin resistance-encoding mecA gene in Staphylococcusspp. and rifampin resistance inMycobacterium tuberculosis. Althoughthese methods may be the future ofdiagnostic testing, they are subject tolimitations. The identification of newmechanisms may be missed as the genesequence is not yet known and primerscannot be generated for them. Molecularmethods are also extremely costly as they require various primers, expensivereagents and skilled individuals to carrythem out.

Phenotypic or synergistic methods area consistent alternative for detectingantibiotic resistance as they are wellestablished and cost-effective. Althoughthey are not as rapid as molecular

Fig 2. Presumptive identification of OXA-48using mastdiscs resistance range.

‘Antibiotic consumptionhas increased by almost40% over the pastdecade, but antibioticdevelopment is almost at a standstill’

PATHOLOGY IN PRACTICE NOVEMBER 2015146

synergy tests are compromised inorganisms that have AmpCs, in whichcase synergy should be sought with afourth-generation cephalosporin andclavulanate, as in the Mastcefepime±clavulanate combination discset (D63C). Alternatively, the Mast AmpC& ESBL Detection Set (D68C) can be usedon all isolates of Enterobacteriaceae toidentify and differentiate AmpC and/orESBL production, which is important assome combinations used to treat ESBLinfections can induce AmpC production.Mast’s ESBL calculator is also availablefor use with D68C, to aid interpretation,and can be downloaded from the Mastwebsite (www.mastgrp.com).

If an equivocal result is generated, theuse of D63C and Mast’s AmpC DetectionSet (D69C) can be used as further work todetermine ESBL and AmpC production.Unlike many other commercially availableproducts, Mast’s AmpC Detection Set detects plasmid and intrinsicchromosomally encoded AmpC enzymes,encompassing inducible, derepressed and hyperproduced strains to provide acomprehensive, reliable and cost-effectivetest.

Regarding carbapenemases, it isrecommended that isolates with decreasedsusceptibility to meropenem, ertapenem or imipenem should be investigated further. For Enterobacteriaceae isolates,

methods, requiring a minimum of 18hours’ incubation, they are the moststandardised method available.

Mast Group provides a comprehensiverange of combination (mastdiscs combi),identification (mastdiscs ID) and AST(mastdiscs AST) discs for the detection ofβ-lactam resistance in Enterobacteriaceae.This range can be easily integrated intothe laboratory to simplify and complementcurrent testing methods.

For ESBL detection, the mastdiscscombi range offers variouscephalosporin/indicator combinations,allowing users to choose betweencefpodoxime±clavulanate, withcefpodoxime being the best generalsubstrate to detect all ESBLs, orcefotaxime±clavulanate andceftazidime±clavulanate, which are goodsubstrates for CTX-M and TEM/SHV-derived enzymes, respectively, but bothmust be used concurrently. These third-generation cephalosporin/clavulanate

MICROBIOLOGY

TABLE 1. DETAILS OF THE MASTDISCS RESISTANCE RANGE.

Code Product Use

D71C(250 tests) CAT-ID Screening of carbapenemase production in Enterobacteriaceae Applicable to EUCAST and CLSI

D70C(50 tests) Carbapenemase Detection Set Confirmation of MBL or KPC production in isolates of Enterobacteriaceae, and differentiation of AmpC + porin loss

D69C(50 tests) AmpC Detection Set Confirmation of AmpC production in isolates of Enterobacteriaceae with either plasmid-acquired or chromosomal AmpC

D68C(50 tests) AmpC & ES�L Detection Set Confirmation of AmpC and/or ESBL production in isolates of Enterobacteriaceae

D52C(50 tests) Extended-spectrum β-lactamase Set Confirmation of ESBL production in isolates of Enterobacteriaceae with Ceftazidime 30 and Ceftazidime 30/ little or no chromosomal de-repressed or inducible AmpC Clavulanic acid 10 (eg Escherichia coli and Klebsiella spp.) Cefotaxime 30 and Cefotaxime 30/ Applicable to CLSI Clavulanic acid 10 Cefpodoxime 30 and Cefpodoxime 30/ Clavulanic acid 10

D67C(50 tests) Extended-spectrum β-lactamase Set Confirmation of ESBL production in isolates of Enterobacteriaceae with (CPD10) little or no chromosomal de-repressed or inducible AmpC Ceftazidime 30 and Ceftazidime 30/ (eg E. coli andKlebsiella spp.) Clavulanic acid 10 Applicable to CLSI, DIN, BSAC Cefotaxime 30 and Cefotaxime 30/ Clavulanic acid 10 Cefpodoxime 10 and Cefpodoxime 10/ Clavulanic acid 1

D62C(150 tests) Cefotaxime 30 and Cefotaxime30/ Confirmation of ESBL production in isolates of Enterobacteriaceae with Clavulanic Acid 10 little or no chromosomal de-repressed or inducible AmpC (eg E. coli and Klebsiella spp.) Applicable to CLSI Must be used in combination with D64C

D63C(150 tests) Cefepime 30 and Cefepime 30/ Confirmation of ESBL production in isolates of Enterobacteriaceae with Clavulanic Acid 10 chromosomal AmpC (eg Enterobacter spp. and Citrobacter spp.)

D64C(150 tests) Ceftazidime 30 and Ceftazidime 30/ Confirmation of ESBL production in isolates of Enterobacteriaceae with Clavulanic Acid 10 little or no chromosomal de-repressed or inducible AmpC (eg E. coli and Klebsiella spp.) Must be used in combination with D62C

D66C(150 tests) Cefpodoxime 10 and Cefpodoxime 10/ Confirmation of ESBL production in isolates of Enterobacteriaceae with Clavulanic Acid 1 little or no chromosomal de-repressed or inducible AmpC (eg E. coli and Klebsiella spp.) Applicable to BSAC and DIN

‘Molecular-based assays have led to a number of importantdiscoveries, including the identification of the methicillinresistance-encoding mecA gene in Staphylococcus spp.’

EUCAST,2 isolates that generate a zonediameter of <11 mm to a temocillin 30 mgdisc are indicative of OXA-48 production.All mastdiscs are compatible with theMast Disc Dispenser, streamlining workflow and aiding rapid integration intoroutine laboratory testing.

Mast also provides specialised MastUri Plates; antibiotic and Identificationmedia in 96-well microtitre plates,compatible with the Mast Uri System, to screen urine samples for the presenceof ESBL, AmpC (MPM-CPD) andcarbapenemase-producing isolates (MPM-CRE). The Mast Uri System is aninnovative, semi-automated laboratorysolution for the microbiological analysis of urine samples, for which Mast wasrecently awarded the OutstandingAchievement award at the Medilink North West and Medilink UK awards. PiP

References1 Nesta. Antibiotics. 2014

(https://longitudeprize.org/challenge/antibiotics).

2 European Committee on AntimicrobialSusceptibility Testing (EUCAST).Antimicrobial susceptibility testing.2013 (www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Disk_test_documents/Manual_v_3.0_EUCAST_Disk_Test.pdf).

3 Health Protection England.Carbapenem resistance: guidance,data, analysis. 2013 (www.hpa.org.uk/Topics/InfectiousDiseases/InfectionsAZ/CarbapenemResistance/GeneralInformation).

4 Centers for Disease Control andPrevention. Antibiotic resistance threatsin the United States. 2013 (www.cdc.gov/drugresistance/threat-report-2013/pdf/ar-threats-2013-508.pdf#page=11).

5 O’Neill J. Antimicrobial resistance.Tackling a crisis for the health andwealth of nations. 2014 (http://amr-review.org/sites/default/files/AMR%20Review%20Paper%20-%20Tackling%20a%20crisis%20for%20the%20health%20and%20wealth%20of%20nations_1.pdf).

6 O’Neill J. Tackling a global health crisis:initial steps. 2015 (http://amr-review.org/sites/default/files/Report-52.15.pdf).

PATHOLOGY IN PRACTICE NOVEMBER 2015 147

ertapenem tends to have the bestsensitivity but poor specificity forcarbapenemase producers, andmeropenem and imipenem have betterspecificity but reduced sensitivity.Accordingly, Mast has developed CAT-ID(D71C) as part of the mastdiscs IDrange, which is a simple screening disccontaining faropenem that does notrequire any measurement or analysis ascarbapenemase production is identifiedvia simple observation. MBL and KPC-producing isolates will display totalresistance to faropenem and no zone is generated around the CAT-ID disc.Furthermore, OXA-48-producing isolatesare easily distinguished from the uniqueformation of a ‘double zone’ around theCAT-ID disc, which features a clear zonewith micro-colonies.

Positive and equivocal results should be confirmed using the MastCarbapenemase Detection Set (D70C),which differentiates between AmpC withporin loss, MBL- and KPC-producingEnterobacteriaceae. D70C is acombination disc test containing anindicator carbapenem alongside a β-lactamase inhibitor. This test can also be used in conjunction with a temocillin 30 mg (TEM30C) disc for presumptiveidentification of OXA-48-producingEnterobacteriaceae. As advised by

MICROBIOLOGY

Erin VaughanMarketing and Technical SupportMast GroupMast HouseDerby Road, Bootle Merseyside L20 1EA Tel: +44 (0)151 933 7277Fax: +44 (0)151 944 1332 Web: www.mastgrp.com