ConnectionsConnections - cmptcmpt.ca/wp-content/uploads/2016/08/Summer-2016.pdf · ty manual and...

HPTA - Biological Safety Officers

In the context of the HPTA every HPTA licence applicant should designate an individual as a biological safety officer (BSO)

The role of the BSO is to promote and monitor compliance with

the provisions of the Act and the regulations This involves

Making sure that every person who conducts controlled activi-

ties authorized by the licence has been properly trained and that their training has been documented

Conducting regular inspections and communicating non com-

pliances so they can be corrected

Assisting in the development and maintenance of the biosafe-

ty manual and standard operating procedures related to bi-osafety and biosecurity

The BSO also communicates with the Minister on behalf of the license holder this involves informing of any inadvertent posses-

sion of a human pathogen or toxin or any deviation from the ex-

pected transport and acquisition of human pathogens or toxins

The individual assigned as BSO must have the following qualifica-

tions

Knowledge of microbiology appropriate to the risks associated

with the controlled activities authorized by the licence

Knowledge of the provisions of the Act regulations and appli-

cable legislation

Knowledge of the applicable biosafety and biosecurity policies

standards and practices appropriate to the risks associated with the controlled activities authorized by the licence

wwwcmptca CMPT Quarterly on -line newsletter Volume 20 Number 2

ConnectionsConnections

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mm

er 20

16

ISSN 1496-3876

INNOVATION EDUCATION QUALITY ASSESSMENT CONTINUAL IMPROVEMENT

IN THIS ISSUE

HPTA - Biological Safety Officershelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip1

Resistance in the Glyopeptide Antimicrobial Class helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip2

Mechanisms of Bacterial Resistance to Aminoglycosides helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip5

News helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip9

Upcoming events helliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphelliphellip10

According to the HPTA regulations the BSO has the power to

require any person who conducts controlled activities authorized

by the licence to provide them with any records that are neces-sary to assist them in carrying out their functions

The intention of this article is to summarize the section about licences of the HPTA For more information please refer to the complete HPTA document

NEXT ISSUE Access to Facilities and Security Clearance

Feature Article

Connections Volume 20 Number Summer 2016 Page 2 of 10

T he glyopeptide antibiotics are

cell-wall active agents that

i n c l u d e g l y c o p e p t i d e s(vancomycin) modified glycopep-

tides (oritivancin and dalbavancin) and lipoglyopeptides (teichoplanin

and televancin) There are a num-ber of other compounds with simi-

lar activity that have not yet been

licensed and will not be discussed here (1)

Mode of Action

The primary mode of action of all these agents is inhibition of

cell-wall synthesis in Gram-positive bacteria There is virtually

no effective activity against gram negative bacterial species Vancomycin is the best studied It binds to the terminal D-

alanine-D-alanine residues of the structural peptides N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAMA) in the

growing cell wall In this way NAGNAMA peptides are prevent-ed from forming together into a structurally strong cell wall and

cross linking of those moieties is also prevented The result is a

weak cell wall that eventually bursts The glycopeptides are bac-tericidal agents

Activity

The antimicrobial activity of the glycopeptides and lipoglycopep-

tides depends on the side chains incorporated into the active

structure of the molecules

Vancomyicn has broad activity against a variety of gram posi-

tive bacteria including Staphylococcus aureus (both MSSA and MRSA) coagulase-negative staphylococci Enterococcus spp

both Streptococcus pyogenes and Streptococcu pneumoniae Bacillus cereus Corynebacterium jeikeium Listeria monocyto-genes and viridans group strepto-

cocci (both penicillin susceptible a n d r e s i s t a n t s t r a i n s )

Teicoplanin has similar activity but is inactive against Enterococcus faecalis Against anaerobic bacte-

ria vancomycin is active against Clostridium difficile Clostridium perfringens and peptostreptococci but has not clinically effec-tive activity against other anaerobic species Dalbavancin and

oritavancin have substantially broader activity against a range of

anaerobes

Modification of the basic structure of the glyopeptide molecule

with addition of glycosyated or acyl side chains or other fatty acids alters the activity of these agents Telavancin is more ac-

tive than vancomycin but less active than oritavancin Dalba-vancin is more active than vancomycin and teicoplanin Each of

the newer derivative agents has additional activity against en-

terococci both vancomycin-susceptible and -resistant strains

The reader should review appropriate literature and summary

data to learn the niche activities for each of these agents Data is available in both EUCAST and CLSI documents for these

agents

Resistance

A number of gram positive bacterial species are intrinsically re-

sistant to vancomycin and teicoplanin These include Leuconos-toc Pediococcus Nocardia Lactobacillus and Erysipelothrix In

these species the cell wall structure does not permit binding of these agents to the growing peptides in the wall

The most extensively studied resistance phenomenon has been in the enterococci Resistance to vancomycin and teichoplanin

has been shown to result from synthesis of modified precursor

molecules with lower affinity for these compounds Six types of resistance have been reported van A B C D E and G The

most common mechanisms observed in clinical strains of entero-cocci are van A B and C

Van A high-level resistance occurs most commonly in E faecium

(MICs of gt 1000 mgL) and less often in E faecalis At the cell wall level the bacteria are able to replace the D-ala-D-ala pep-

tide with D-ala-D-lac This results in a loss of a critical hydrogen bond for vancomycin and resistance functionally is complete

The resistance genes are carried on a transposon that encodes genes for seven polypeptides which are involved in the develop-

ment of resistance van R and S regulate gene expression for

resistance van H A and X confer resistance to vancomcyin and teicoplanin van Y and Z are accessory proteins that are not di-

rectly involved in resistance van X (a dipeptidase) is required for complete resistance and van Y (a carboxypeptidase) is capa-

ble of cleaving late cell wall development by cleaving D-ala in

those cells The mechanism for van Z involvement is not com-pletely known This high level resistance is inducible by vanco-

mycin It is plasmid mediated and transferable

Van B resistance is lower level

(MICs 8 ndash 64 mgL) occurs in

both E faecium and E faecalis and confers resistance to vanco-

mycin but not teicoplanin Van B determinants are also found on

transposons both on plasmids and chromosomally Mutations that

arise in the van gene clusters in enterococci in these strains re-

sults in impaired phosphorylation of van R and S and result in the low and variable MICs observed in these strains

Van C resistance occurs in E gallinarum E cassiliflaveus and E flavescens These species are intrinsically resistant to low con-

centrations of vancomycin but are susceptible to teicoplanin

For these species three genes van C-1 van C-2 and van C have been described These genes are constitutively expressed

and chromosomally encoded They are not transferable on plas-mids and therefore are not a concern from the infection pre-

vention and control perspective

ldquoResistance to vancomycin and teicoplanin has been shown to result from synthesis of modified precursor molecules with lower affinity for these compoundsrdquo

Resistance in the Glycopeptide Antimicrobial Class

By Robert P Rennie

Connections Volume 20 Number 2 Summer 2016 Page 3 of 10


The derivative glycopeptides have different mechanisms of ac-

tivity (eg oritavancin acts on lipid formation and transglycosyl-ation instead of peptide formation) In this way for example

vancomycin-resistant enterococci remain susceptible to these compounds with MICs of le 1-2 mg For Van A resistant E fae-cium (MICsgt 256 mgL) the MIC90 for oritavancin is 025 mgL

Resistance to vancomycin has also been observed in S aureus primarily in methicillin-resistant strains (MRSA) MRSA strains

with decreased susceptibility to vancomycin (vancomycin inter-mediate-resistant S aureus (VISA or GISA) and more recently

with high-level vancomycin resistance (vancomycin-resistant S aureus (VRSA) have been described in the clinical literature The

rare VRSA strains carry transposon Tn1546 acquired from van-

comycin-resistant E faecalis which is known to alter cell wall structure and metabolism but the resistance mechanisms in

GISA isolates are less well defined These strains are described as hetero-resistant Decreased susceptibility is induced in the

presence of vancomycin and teichoic acid in the cell wall of

these isolates increases The thicker cell wall (clearly observed by electron microscopy) results in reduced affinity of vancomy-

cin for the growing cell wall and an increase in MIC to 4 ndash 8 mg

L True VRSA strains have high MICs gt 16 - 32 mgL (23)

These MRSA strains have remained susceptible to newer deriva-tive glycopeptides

Efficacy

The more recently developed glycopeptides agents offer addi-

tional parameters that make them potentially more effective for

gram positive bacterial infections than vancomycin They have broader activity less resistance considerably longer half-lives

(eg the half-life for dalbavancin is approximately one week) and are well tolerated

In vitro laboratory testing

There are some issues with in-vitro laboratory testing that may

result in the appearance of false resistance Media effects and

inoculum concentration are known to affect MICs particularly for teicoplanin and to a lesser degree for vancomycin For example

it has been shown that the addition of bile salts enhances the appearance of van B strains of enterococci with lower MICs For

the newer lipoglycopetide agents it has been shown that in

broth dilution tests addition of polysorbate (Tween 80) is neces-sary to obtain an accurate MIC In studies without incorporation

Reprinted by permission from Macmillan Publishers Ltd NATURE CHEMICAL BIOLOGY ndash Antibiotics Vancomycin sensing Arthur M Nature Chemi-

cal Biology 6 313-315 copyright 2010

Figure 1 Activity of vancomycin and mechanism of resistance



References

Glycopeptides and Lipoglycopeptides In Antimicrobial Agents Antbacterials and Antifungals 2005 Bryskier A (ed) ASM

Press American Society for MicrobiologyWashington DC Chap-ter 31pp 880-905

Gardete S Tomasz A 2014 Mechanisms of vancomycin re-

sistance in Staphylococcus aureus 2014 J Clin Invest124 2836-40

Webster D Rennie RP Brosnikoff CL Chui L Brown C2007 Methicillin-resistant Staphylococcus aureus with reduced suscep-

tibility to vancomycin in Canada

Diagn Microbiol Infect Dis57177-81

Rennie RP Koeth L Jones RN Fritsche TR Knapp CC Killian SB

Goldstein BP 2007 Factors influencing broth microdilution anti-microbial susceptibility test results for dalbavancin a new glyco-

peptide agentJ Clin Microbiol 45 3151-4

Arthur M 2010 Antibiotics Vancomycin sensing Nat Chem Biol

6 5 313-315

of that agent MICs are as much as 8 fold greater resulting in

apparent higher levels of resistance that do not correlate with clinical outcome (4) These agents are large molecules (like van-

comycin) and disc diffusion studies have resulted in relatively small zones of inhibition even in susceptible isolates For those

reasons dilution testing has been more accurate for this antimi-

crobial group

Testing for vancomycin resistance in S aureus is difficult and

usually requires population analysis studies to separate strains with reduced susceptibility (MICs of 2 ndash 3 mgL) from GISA and

VRSA strains Automated systems may overcall resistance MICs by gradient diffusion techniques are usually lower and may as-

sist in separating isolates with only decreased susceptibility

Summary

Antimicrobial activity in the glycopeptide group of antibacterial

agents is directed against cell wall components of gram-positive bacterial species Resistance occurs either through plasmid

transfer or is constitutive depending on the mechanism and

species Testing for resistance is these bacterial species requires special attention to media inoculum and to the properties of

each of the agents within the group

We would like to thank Dr Rennie for his contribution to our newsletter

Dr Rennie is a Clinical Microbiologist for the Alberta Health Services He is Chair of the CLSI Subcommittee on Cul-ture Media member of the Canadian Standards Association ISO Z252TC212 Committee on Quality Laboratory Man-

agement and member (former Chair) of the Clinical Microbiology Proficiency Testing (CMPT) Microbiology Subcom-mittee

This meeting will be valuable for anyone interested in the study of customer satisfaction regardless of industry It will be of particular interest to people engaged in medical laboratory sciences including

Administrators Pathologists Quality Specialists Accreditation

Technologists Residents Graduate Students Phlebotomists Nurses

httpconference2016polqmca


Feature Article

MECHANISM OF ACTION AND PHARMACODYNAMICS

At pH 74 aminoglycosides have a high positive charge (cationic) which contributes to both their antimicrobial and tox-

icity properties2 The positively charged aminoglycoside forms a bond with the negatively charged (anionic) bacterial cell mem-

brane In order to reach the intracellular ribosomal binding site

an aerobic energy-dependent process (phase I) is needed to allow a fraction of the drug attached to the bacterium to be

transported through the inner bacterial cell membrane This aer-obic process explains why anaerobic organisms have intrinsic

resistance to aminoglycosides and why there is diminished ac-tivity to microorganisms growing in an anaerobic environment

such as abscesses Some facultative anaerobes (such as entero-

cocci or other small colony streptococcal variants) also have de-ficient energy-dependent uptake and therefore are resistant to

low concentrations of aminoglycosides

Once the cell membrane has been penetrated binding of the

aminoglycoside with the ribosomal 30S subunit using hydrogen

bonds between multiple amino and hydroxyl groups produce a tightly bound complex The aminoglycoside-bound 30S subunits

become unavailable for translation of mRNA during protein syn-thesis and cause misreading of the genetic code with resultant

production of non-functional proteins (missense and nonsense mutations) The non-functional protein production and displace-

ment of Mg2+ and Ca2+ ions cause increased cell membrane per-

meability allowing a higher concentration of aminoglycosides to permeate the cell (phase II) which leads to cell death9 This

concentration-dependent killing of aminoglycosides is one of the main characteristics of their antibacterial activity Unlike other

agents that inhibit microbial protein synthesis aminoglycosides

are bactericidal rather than bacteriostatic2

With the concentration-dependant killing attribute there is in-

creased uptake and synergistic effect when aminoglycoside ther-apy is combined with cell wall-active antimicrobials such as β-

lactams or glycopeptides This synergy between aminoglyco-

sides and cell-wall active drugs is effective for treating entero-cocci viridans streptococci methicillin susceptible Staphylococ-cus aureus and Pseudomonas aeruginosa

A third antibacterial activity of note for aminoglycosides is the

suppression of bacterial growth after short antimicrobial expo-sure (post-antibiotic effect or PAE) In vitro studies have demon-

strated that there is suppression of bacterial growth after short

antimicrobial exposure (post-antibiotic effect or PAE) at amino-glycoside levels less than the MIC and furthermore that the du-

ration of the PAE increases with higher doses2

PHARMACOKINETICS

Aminoglycosides are not metabolized and are excreted virtually

unchanged by the kidney via glomerular filtration Because of their positive electrical charge at physiologic pH they have oto-

toxic and nephrotoxic potential2 Gastrointestinal absorption of

A minoglycoside antibiotics are

derived from compounds pro-duced by a variety of soil actinomy-

cetes and have a similar structure

consisting of two or more amino sugars linked by glycosidic bonds to

an aminocyclitol ring (streptidine or 2-deoxy-streptamine)1 The com-

monly used agents in Canada include

gentamicin tobramycin streptomy-cin and amikacin (and topical neo-

mycin) Other aminoglycosides in use elsewhere or of historical interest include kanamycin and netilmicin aminogylcosides are

bactericidal agents that function by inhibiting bacterial protein synthesis they have a narrow therapeutic-to-toxic ratio and are

often used in the treatment of serious aerobic gram negative

infections2 Some have activity against mycobacteria and others are used in combination with β-lactams for synergy Bacterial

resistance to aminoglycosides can be attributed to three mecha-nisms (i) decreased drug uptake whether by decreased cell

permeability or expulsion by efflux pumps (ii) modification of

the target 16S ribosomal RNA (rRNA) binding site by mutation or methylation and (iii) enzymatic deactivation of the aminoglyco-

side through expression of aminoglycoside-modifying enzymes (AMEs)4

ORIGIN

Naturally occurring aminoglycosides are derived from the actino-

mycete Micromonospora spp (gentamicin) or from Streptomyces spp (kanamycin neomycin streptomycin and tobramycin) whereas amikacin is the semisynthetic derivative of kanamycin

The difference in spelling denotes the origin of the antibiotic -micin indicates derivation from Micromonospora spp and ndashmycin

from Streptomyces spp2 Streptomycin was the first antibiotic in

the aminoglycoside family to be utilized (in the 1940s) and was extrapolated from a strain of Streptomyces griseus

Mechanisms of bacterial resistance to aminoglycosides

By Lorraine Campbell

Figure 1 Streptomyces species Image courtesy of Public Health Image Library


Resistance to Aminoglycosides

AMINOGLYCOSIDE RESISTANCE MECHANISMS

Efflux pumps transport antibiotics from within the cell into the external environment rendering the antibiotic useless The in-

ducible RND-type pump possessed by P aeruginosa and AcrD multidrug efflux transporter in E coli are both capable of eject-

ing aminoglycosides out of the bacterial cell16

Ribosomal mutations of the ribosomal proteins and 16S rRNA along with enzymatic methylation of the rRNA will interfere with

the aminoglycosides ability to bind to the rRNA therefore confer-ring resistance The rRNA methyltransferase enzymes (RmtA

RmtB RmtC RmtD RmtE and RmtF) produce broad-spectrum resistance to the aminoglycosides and are being discovered

more frequently world-wide on plasmids with broad-spectrum b-

lactamases like CTX-M NDM and KPC3

By far aminoglycoside-modifying enzymes (AMEs) are the most

common cause of aminoglycoside resistance AMEs catalyze the covalent modification of aminoglycosides as they transport

across the cytoplasmic membrane by modifying the amino or

hydroxyl groups3 N-acetyltransferases (AAC) modify the ndashNH2

(amino) group by N-acetylation Hydroxyl groups are modified

by either O-nucleotidyltranferases (ANT) by O-nucleotidylation or O-phosphotranferases (APH) by O-phosphorylation The acet-

ylation adenylation or phosphorylation of the aminoglycoside reduces drug binding to the ribosome which results in high lev-

els of resistance it also negates the synergistic activity of the

aminoglycoside with b-lactams The level of resistance can also depend on the affinity of the specific aminoglycoside to the

AME the higher the affinity the less amount of enzyme is need-ed to inactivate the aminoglycoside AMEs are highly mobile and

may be coded on the chromosome or spread by genes on plas-

mids andor transposons As a consequence there is a broad range of bacteria that can support enzymatic resistance to ami-

noglycosides7

Each enzyme is described by its class (AAC ANT or APH) a

number in parentheses signifying the location of the modifica-

tion of the drug and a Roman numeral indicating a unique ami-noglycoside resistance phenotype as they can differ greatly

Currently there are seven major clinically relevant phosphotrans-ferases four nucleotidyltransferases and four acetyltranferases

(Table 2)3

aminoglycosides is minimal due in part to their high polarity so

they are normally administered parenterally either intravenously or intramuscularly After intravenous administration aminoglyco-

sides are freely distributed in the vascular and extracellular space but have poor penetration into cerebrospinal fluid (CSF)

and vitreous fluid25 Thus aminoglycosides may rarely need to

be administered intrathecally or intravitreally in select clinical circumstances

CLINICAL INDICATIONS

Aminoglycosides are often utilized for empirical treatment of se-

vere infections suspected to be caused by aerobic gram negative bacilli especially when used in combination with b-lactam or

vancomycin therapy Gentamicin and tobramycin are recom-

mended by CLSI as first-line drugs to test and report for Entero-bacteriaceae and P aeruginosa as well as testing amikacin for

selective reporting8 Gentamicin and tobramycin share similar activity and deactivation by AMEs whereas amikacin shows

more resistance to many of the AMEs thus making it a useful

option in very resistant bacteria1

They can be used as definitive therapies for gram negative infec-

tions especially pyelonephritis once the susceptibility testing is reported Because of the risk of toxicity use of aminoglycosides

has become less common as effective safer antimicrobials have been introduced to clinical practice

As discussed above enterococci are inherently resistant to low

levels of aminoglycosides However their synergistic activity with cell wall-active agents makes them essential to curing

endovascular infections (ie endocarditis) caused by Enterococ-cus spp2 Laboratory testing is done to rule out high level re-

sistance by using extremely high levels of drug (eg for gen-

tamicin a 120microg disc or a 500 microgmL broth microdilution well) The aminoglycosides normally used for this purpose include gen-

tamicin and streptomycin

Streptomycin has seen less use in recent years but it is still used

in multidrug therapies to treat M tuberculosis infections espe-

cially when resistance to first-line agents is detected It remains the drug of choice for rare infections such as Yersinia pestis and

Francisella tularensis1

Resistance type Resistance mechanism Aminoglyco-side inactivated

Bacteria affected

Decreased uptake Changes in outer membrane permeability All P aeruginosa

Decreased uptake Efflux systems such as

Resistance nodulation cell division (RND) Major facilitator superfamily (MFS)

All wide range

Modification of the ribosome Point mutations in ribosomal protein S12 and in the 16S rRNA

Streptomycin Mycobacterium tuberculosis

Modification of the ribosome 16S rRNA point mutation Amikacin Mycobacterium abscessus and Mycobacterium chelonae

Modification of the ribosome Methylation of the 16S rRNA by the enzyme rRNA methyltransferase (Rmt)

All Gram negative bacilli

Table 1 Aminoglycoside resistance mechanisms ndash Decreased uptake and modification of ribosome



Resistance type Enzyme subclass Aminoglycoside inactivated Bacteria affected

Phosphorylation Enzymes (APH)

Enzyme inactivation via phosphory-lation of the aminoglycoside

APH(2rdquo) Kanamycin Tobramycin Gentamicin

Staphylococci Streptococci Enterococci

APH(3rsquo) Kanamycin Neomycin Amikacin

Enterobacteriaceae Pseudomonas Staphylococci Streptococci Enterococci Corynebacterium

APH(6) Streptomycin Gram-negative organisms

Acetylation Enzymes (AAC)


Enzyme inactivation via acetylation of the aminoglycoside

AAC(2rsquo) Gentamicin Tobramycin

Providencia-Proteus Mycobacterium

AAC(3rsquo) Kanamycin Tobramycin Gentamicin

Enterobacteriaceae Pseudomonas

AAC(6rsquo) Kanamycin Tobramycin Amikacin

Enterobacteriaceae Pseudomonas Staphylococcus Enterococcus

Adenylation Enzymes (ANT)


Enzyme inactivation via adenylation of the aminoglycoside

ANT(2rdquo) Kanamycin Tobramycin Gentamicin


ANT(3rdquo) Streptomycin Enterococcus Pseudomonas

ANT(4rsquo) Kanamycin Tobramycin Amikacin

Staphylococcus Enterococcus

ANT(6rsquo) Streptomycin Wide spread amongst Gram positive bacteria

Bifunctional Enzymes


AAC(6rsquo)APH(2rdquo)

Gentamicin Tobramycin Amikacin Kanamycin Arbekacin

Staphylococcus Enterococci

Enzymatic inactivation

AAC(6rsquo)-Ib cr

Gentamicin Kanamycin Tobramycin Fluoroquinolone

Enterobacteriaceae

Table 2 Aminoglycoside resistance mechanisms ndash Aminoglycoside-modifying enzymes

Source Table adapted from Mandell Douglas and Bennett 2015 Molecular Mechanisms of Antibiotic Resistance in Bacteria Principles and Practice of Infectious Diseases Eighth Edition Elsevier Saunders Philadelphia PA P242

Recently reports have described bi-functional enzymes that

modify the structure of an entirely different class of antimicrobial agent (ciprofloxacin) as well as aminoglycosides One such en-

zyme is designated as AAC(6rsquo)-Ib-cr which acetylates kanamy-cin gentamicin and tobramycin as well as the piperazinyl side

group of ciprofloxacin3 Another recent development was the

discovery of the bi-functional enzyme AAC(6rsquo)APH(2rdquo) with two functioning active sites (one for acetylation and the other for

phosphorylation of aminoglycosides) This bi-functional enzyme is now readily seen in staphylococci and enterococci on a com-

mon transposon Tn4001 from transferable plasmids or on the chromosome and confers high levels of resistance to the amino-

glycosides3

THE FUTURE OF AMINOGLYCOSIDES

Efforts to keep aminoglycosides as useful weapons in the arsenal

against bacterial infectious diseases include development of ami-noglycosides that are more unyielding to AMEs and the develop-

ment of inhibitors of AMEs79 Antibiotic stewardship is vital to the

future of all antibiotic efficacy Measures include limiting use of antibiotics only when indicated education for patients about the

importance of finishing their prescription and de-escalation or narrowing antibiotic therapy from an empirical broad spectrum

antimicrobial to a pathogen specific narrow spectrum drug when the laboratory susceptibility results are available

There have been several studies with promising results of in

vitro activity of plazomicin a new generation aminoglycoside (formerly ACHN-490) against multi-drug resistant clinical isolates

of K pneumoniae E coli Enterobacter spp and even MRSA 10 11 Plazomicin does not appear to be compromised by most

clinically relevant AMEs but does appear to be affected by me-

thyltransferases

We would like to thank Ms Lorraine Campbell for her

contribution to our newsletter

Ms Lorraine Campbell is staff at the Microbiology Labor-

atory Calgary Laboratory Services Calgary AB and a member of the Clinical Microbiology Proficiency Testing

(CMPT) Microbiology Subcommittee



References

1 Jorgensen JH et al 2015 Antibacterial Agents and Suscepti-bility Test Methods Manual of Clinical Microbiology Eleventh

ed ASM Press Washington DC pp 1180-1182

2 Mandell Douglas and Bennett 2015 Aminoglycosides Princi-

ples and Practice of Infectious Diseases Eighth Edition Else-

vier Saunders Philadelphia PA pp 224-233

3 Mandell Douglas and Bennett 2015 Molecular Mechanisms of

Antibiotic Resistance in Bacteria Principles and Practice of In-fectious Diseases Eighth Edition Elsevier Saunders Philadel-

phia PA pp 235-251

4 Jorgensen JH et al 2015 Mechanisms of Resistance to Anti-

bacterial Agents Manual of Clinical Microbiology Eleventh ed

ASM Press Washington DC Pp 1212-1245

5 Mandell Douglas and Bennett 2015 Principles of Anti-

infective Therapy Principles and Practice of Infectious Diseas-es Eighth Edition Elsevier Saunders Philadelphia PA pp 224-

234

6 Webber MA Piddock LJV The importance of efflux pumps in bacterial antibiotic resistance 2003 Journal of Antimicrobial Chemotherapy 51 9-11

7 Ramirez MS Tolmasky ME 2010 Aminoglycoside Modifying

Enzymes Drug Resist Update Dec 13(6) 151-171

8 CLSI Performance Standards for Antimicrobial Susceptibility Testing Twenty-Fifth Informational Supplement CLSI docu-

ment M100-S25 Wayne PA Clinical and Laboratory Stand-ards Institute 2015

9 Vakulenko SB Mobashery S Versatility of Aminoglycosides and Prospects for Their Future 2003 Clinical Microbiology

Reviews 61 430-450

10Walkty A et al In Vitro Activity of Plazomicin against 5015 Gram-Negative and Gram-Positive Clinical Isolates Obtained

from Patients in Canadian Hospitals as Part of the CANWARD Study 2011-2012 2014 Antimicrobial Agents and Chemother-

apy May 58(5) 2554-2563

11Galani I et al Activity of Plazomicin (ACHN-490) against MDR clinical isolates of Klebsiella pneumonia Escherichia coli and

Enterobacter spp From Athens Greece 2012 Journal of Chemotherapy (Florence Italy) Aug 24(4) 191-194


News

Changes on Grading of Reporting Errors

Identifier Errors

The use of incorrect identifiers to report results will not affect the

grade of the different challenge components

However a reporting error point will be given for each challenge

challenge component reported using the incorrect identifier

These points can be used by the laboratory to track reporting errors

Laboratories will be notified of identification errors if applicable on

the survey result letters

CMPT Annual General Meeting - Open House

CMPT will be holding its Annual General Meeting on Tuesday

October 4 2016 (9am - 4pm) at the Holiday Inn Vancouver Cen-

tre

The topics on discussion at the AGM include CMPT activities over

the last year how we and laboratories have performed and fu-

ture plans Guest speakers will present on related topics

There will be reserved seating available for LIMITED number of

laboratory observers to attend and participate in the meeting

Although attendance to the CMPT AGM is free of charge regis-

tration is required (please register before September 02 2016)

Contact Michael Noble (mnoblemailubcca ) or Esther

Kwok (cmptpathubcca) to register for the AGM and please

include in your query both your laboratorys name and number

We are getting close to the end of our 2015-2016 PD Course

There is time until the end of September to complete all quiz-zes

Please remember that certificates of completion will be given only to those participants that complete all modules (with pass-

ing grades) of a particular category or all categories if they

have completed all the quizzes

Clinical Microbiology Module 4 has been recently released

Parasitology Module 3 will be released midend of August

For more information check the coursersquos website or contact the course administrator restellimailubcca

CMPT Professional Development course

ABOUT CONNECTIONS

ldquoConnectionsrdquo is published quarterly by CMPT and is aimed at the Microbi-ology staff Editor Veronica Restelli Contact Connections By mail Room G408 2211 Wesbrook Mall Vancouver BC V6T 2B5 Canada By phone 604ndash 827-1754 By fax 604-827-1338 By email restellimailubcca Connections is available online wwwcmptcanewsletter_connectionshtml We want to hear from you Please fol-low the link to submit questions sug-gestions articles information about events etc wwwcmptcanewsletter_bulletinnews_submissionshtm

Get Connected

Connections Volume 20 Number 2 Summer 2016 0 of 10

September 2016

ESCMIDASM Conference on Drug Development to Meet the Challenge of An-timicrobial Resistance

September 21 - 23 2016 Vienna Austria

More info httpswwwescmidorgresearch_projectsescmid_conferencesescmidasm_conference

October 2016

CMPT Annual General Meeting

October 04 2016 Vancouver BC

More info infocmptca

POLQM Fall Conference - Customer Satisfaction and the Medical Laboratory

October 5 2016 UBC Life Sciences Centre Vancouver BC

More info httpconference2016polqmca

April 2017

27th European Congress of Clinical Microbiology and Infectious Diseases April 22 - 25 2017 Vienna Austria

More info httpwwweccmidorg

Upcoming Events

Feature Article

Connections Volume 20 Number Summer 2016 of 10

T he glyopeptide antibiotics are

cell-wall active agents that

i n c l u d e g l y c o p e p t i d e s(vancomycin) modified glycopep-

tides (oritivancin and dalbavancin) and lipoglyopeptides (teichoplanin

and televancin) There are a num-ber of other compounds with simi-

lar activity that have not yet been

licensed and will not be discussed here (1)

Mode of Action

The primary mode of action of all these agents is inhibition of

cell-wall synthesis in Gram-positive bacteria There is virtually

no effective activity against gram negative bacterial species Vancomycin is the best studied It binds to the terminal D-

alanine-D-alanine residues of the structural peptides N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAMA) in the

growing cell wall In this way NAGNAMA peptides are prevent-ed from forming together into a structurally strong cell wall and

cross linking of those moieties is also prevented The result is a

weak cell wall that eventually bursts The glycopeptides are bac-tericidal agents

Activity

The antimicrobial activity of the glycopeptides and lipoglycopep-

tides depends on the side chains incorporated into the active

structure of the molecules

Vancomyicn has broad activity against a variety of gram posi-

tive bacteria including Staphylococcus aureus (both MSSA and MRSA) coagulase-negative staphylococci Enterococcus spp

both Streptococcus pyogenes and Streptococcu pneumoniae Bacillus cereus Corynebacterium jeikeium Listeria monocyto-genes and viridans group strepto-

cocci (both penicillin susceptible a n d r e s i s t a n t s t r a i n s )

Teicoplanin has similar activity but is inactive against Enterococcus faecalis Against anaerobic bacte-

ria vancomycin is active against Clostridium difficile Clostridium perfringens and peptostreptococci but has not clinically effec-tive activity against other anaerobic species Dalbavancin and

oritavancin have substantially broader activity against a range of

anaerobes

Modification of the basic structure of the glyopeptide molecule

with addition of glycosyated or acyl side chains or other fatty acids alters the activity of these agents Telavancin is more ac-

tive than vancomycin but less active than oritavancin Dalba-vancin is more active than vancomycin and teicoplanin Each of

the newer derivative agents has additional activity against en-

terococci both vancomycin-susceptible and -resistant strains

The reader should review appropriate literature and summary

data to learn the niche activities for each of these agents Data is available in both EUCAST and CLSI documents for these

agents

Resistance

A number of gram positive bacterial species are intrinsically re-

sistant to vancomycin and teicoplanin These include Leuconos-toc Pediococcus Nocardia Lactobacillus and Erysipelothrix In

these species the cell wall structure does not permit binding of these agents to the growing peptides in the wall

The most extensively studied resistance phenomenon has been in the enterococci Resistance to vancomycin and teichoplanin

has been shown to result from synthesis of modified precursor

molecules with lower affinity for these compounds Six types of resistance have been reported van A B C D E and G The

most common mechanisms observed in clinical strains of entero-cocci are van A B and C

Van A high-level resistance occurs most commonly in E faecium

(MICs of gt 1000 mgL) and less often in E faecalis At the cell wall level the bacteria are able to replace the D-ala-D-ala pep-

tide with D-ala-D-lac This results in a loss of a critical hydrogen bond for vancomycin and resistance functionally is complete

The resistance genes are carried on a transposon that encodes genes for seven polypeptides which are involved in the develop-

ment of resistance van R and S regulate gene expression for

resistance van H A and X confer resistance to vancomcyin and teicoplanin van Y and Z are accessory proteins that are not di-

rectly involved in resistance van X (a dipeptidase) is required for complete resistance and van Y (a carboxypeptidase) is capa-

ble of cleaving late cell wall development by cleaving D-ala in

those cells The mechanism for van Z involvement is not com-pletely known This high level resistance is inducible by vanco-

mycin It is plasmid mediated and transferable

Van B resistance is lower level

(MICs 8 ndash 64 mgL) occurs in

both E faecium and E faecalis and confers resistance to vanco-

mycin but not teicoplanin Van B determinants are also found on

transposons both on plasmids and chromosomally Mutations that

arise in the van gene clusters in enterococci in these strains re-

sults in impaired phosphorylation of van R and S and result in the low and variable MICs observed in these strains

Van C resistance occurs in E gallinarum E cassiliflaveus and E flavescens These species are intrinsically resistant to low con-

centrations of vancomycin but are susceptible to teicoplanin

For these species three genes van C-1 van C-2 and van C have been described These genes are constitutively expressed

and chromosomally encoded They are not transferable on plas-mids and therefore are not a concern from the infection pre-

vention and control perspective

ldquoResistance to vancomycin and teicoplanin has been shown to result from synthesis of modified precursor molecules with lower affinity for these compoundsrdquo


By Robert P Rennie

















Efficacy

















References












6 5 313-315





crobial group





Summary














Feature Article






























PHARMACOKINETICS



















ORIGIN
































noglycosides7





(Table 2)3




























Bacteria affected




All wide range









































AAC(6rsquo)-Ib cr


Enterobacteriaceae










glycosides3












thyltransferases








References








phia PA pp 235-251






234







Reviews 61 430-450



apy May 58(5) 2554-2563




News


Identifier Errors











tre




















ABOUT CONNECTIONS


Get Connected


September 2016




October 2016







April 2017



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Connections Volume 20 Number 2 Summer 2016 of 10
















Efficacy

















References












6 5 313-315





crobial group





Summary














Feature Article






























PHARMACOKINETICS



















ORIGIN
































noglycosides7





(Table 2)3




























Bacteria affected




All wide range









































AAC(6rsquo)-Ib cr


Enterobacteriaceae










glycosides3












thyltransferases








References








phia PA pp 235-251






234







Reviews 61 430-450



apy May 58(5) 2554-2563




News


Identifier Errors











tre




















ABOUT CONNECTIONS


Get Connected


September 2016




October 2016







April 2017



Upcoming Events



References












6 5 313-315





crobial group





Summary














Feature Article






























PHARMACOKINETICS



















ORIGIN
































noglycosides7





(Table 2)3




























Bacteria affected




All wide range









































AAC(6rsquo)-Ib cr


Enterobacteriaceae










glycosides3












thyltransferases








References








phia PA pp 235-251






234







Reviews 61 430-450



apy May 58(5) 2554-2563




News


Identifier Errors











tre




















ABOUT CONNECTIONS


Get Connected


September 2016




October 2016







April 2017



Upcoming Events


Feature Article






























PHARMACOKINETICS



















ORIGIN
































noglycosides7





(Table 2)3




























Bacteria affected




All wide range









































AAC(6rsquo)-Ib cr


Enterobacteriaceae










glycosides3












thyltransferases








References








phia PA pp 235-251






234







Reviews 61 430-450



apy May 58(5) 2554-2563




News


Identifier Errors











tre




















ABOUT CONNECTIONS


Get Connected


September 2016




October 2016







April 2017



Upcoming Events
























noglycosides7





(Table 2)3




























Bacteria affected




All wide range









































AAC(6rsquo)-Ib cr


Enterobacteriaceae










glycosides3












thyltransferases








References








phia PA pp 235-251






234







Reviews 61 430-450



apy May 58(5) 2554-2563




News


Identifier Errors











tre




















ABOUT CONNECTIONS


Get Connected


September 2016




October 2016







April 2017



Upcoming Events



































AAC(6rsquo)-Ib cr


Enterobacteriaceae










glycosides3












thyltransferases








References








phia PA pp 235-251






234







Reviews 61 430-450



apy May 58(5) 2554-2563




News


Identifier Errors











tre




















ABOUT CONNECTIONS


Get Connected


September 2016




October 2016







April 2017



Upcoming Events








glycosides3












thyltransferases








References








phia PA pp 235-251






234







Reviews 61 430-450



apy May 58(5) 2554-2563




News


Identifier Errors











tre




















ABOUT CONNECTIONS


Get Connected


September 2016




October 2016







April 2017



Upcoming Events


News


Identifier Errors











tre




















ABOUT CONNECTIONS


Get Connected


September 2016




October 2016







April 2017



Upcoming Events

ABOUT CONNECTIONS


Get Connected


September 2016




October 2016







April 2017



Upcoming Events

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