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Daniel Garang Kuir. BBioMedSci, USQ M App Sci (MedSci), RMIT.

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Presentation on theme: "Daniel Garang Kuir. BBioMedSci, USQ M App Sci (MedSci), RMIT."— Presentation transcript:

1 Daniel Garang Kuir. BBioMedSci, USQ M App Sci (MedSci), RMIT

2  Members of Enterobacteriaceae family are a heterogeneous group of gram negative bacteria.  Are part of human’s normal enteric flora.  Are also abundantly distributed in nature.  Include some prominent, often opportunistic, human pathogens;  Such as E. coli (e.g uropathogenic E. coli), Klebsiella spp, Enterobacter spp, Citrobacter spp, Salmonella spp, Shigella spp, Yersinia pestis, Serratia marcescens, Proteus spp, Morganella spp, & Providencia spp.  Majority are often expediently termed as the “ESCPPM” organisms – which stands for Enterobacter spp, Serratia spp, Citrobacter freundii, Proteus vulgaris & penneri, Providencia spp, & Morganella morganii.  Several members of this group are ESBL - &/or AmpC- producers.  K. pneumoniae & E. coli are major producers of ESBLs in this group of gram negative bacteria.

3  Production of β-lactamases in Enterobacteriaceae is a common mechanism of antimicrobial resistance.  These β-lactamases include the novel β-lactamases such as ESBLs, AmpC…etc, & others such as; ◦ Penicillinase, cephalosporinase, broad-spectrum, extended-spectrum, carbapenemase.  AmpC β-lactamases are chromosomally encoded cephalosporinases (chromosomal bla genes).  AmpC are expressed in many Enterobacteriaceae and other organisms.  AmpC induce, by constitutive hyperproduction or mutation, wide-ranging resistance to first-, second-, and third- generation cephalosporins, most penicillins, and beta- lactam/beta-lactam-inhibitor (BL/BLI) combinations.

4  Production of novel β-lactamases e.g. ESBLs, AmpC;  In tandem with production of β-lactamases, Enterobacteriaceae employ other mechanisms of resistance such as; ◦ enzymatic inactivation; ◦ efflux pumps; ◦ outer membrane porin loss; ◦ target modifications; ◦ transfer or acquisition of new genetic material, or ◦ mutations – ESBLs are essentially derivative enzymes acquired through mutations - substitution or deletion of amino acids - in progenitor β- lactamases (e.g TEM, SHV or CTX-M).

5  ESBLs are novel β-lactamases - are newer β-lactamases of pathogenic gram negative bacteria (esp. Enterobacteriaceae family). ◦ These novel β-lactamases also include;  Plasmid-mediated AmpC β-lactamases;  Carbapenem-hydrolysing β-lactamases (e.g. Klebsiella pneumoniae carbapenemases (KPC));  Β-lactamases with reduced sensitivity to β-lactamases inhibitors  Definition: ESBLs are bacterial enzymes capable of hydrolysing and thus conferring resistance to all penicillins, first-, second-, & third- generation cephalosporins, and aztreonam.  And are inhibited by β-lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam.  ESBLs are plasmid-mediated enzymes that confer multi-drug resistance to gram negative bacteria.  ESBLs may be co-expressed &/or co-transmitted with chromosomally- encoded AmpC β-lactamases – thus presence of ESBLs may be masked by AmpC.

6  ESBLs hydrolyse all β-lactam antibiotics – penicillins and cephalosporins.  β-lactamases possess either a serine moiety or a zinc atom in the active site,  Either of which is vital for hydrolysis of the β-lactam ring of a β–lactam antibiotic.  ESBLs are diverse, quickly evolving & therapeutically difficulty to eradicate.  ESBL production in Enterobacteriaceae also render them resistant to other major classes of antibiotics such as; ◦ Fluoroquinolones (e.g. ciprofloxacin, norfloxacin), ◦ Aminoglycosides (e.g. gentamicin, tobramycin, amikacin) ◦ Tetracyclines (e.g. tetracycline) ◦ Trimethroprims-sulfamethoxazole (Cotrimoxazole) ◦ Other antibiotic classes NB : β-lactamase production, co-expression of ESBL &/or AmpC, carriage of other resistance gene on the same plasmid account for multidrug resistance in this group of bacteria.  ESBL-mediated extensive antimicrobial resistance poses public health risks.  ESBL-producing Enterobacteriaceae are essentially multidrug resistant bacteria.

7 Source: Rosário NA, Grumach AS. Allergy to beta-lactams in paediatrics: a practical approach. J Pediatr (Rio J). 2006;82(5 Suppl):S181-8.

8 Source: Partridge, S. (2014). Movement of resistance genes in hospitals. Microbiology Australia.

9  ESBL-producing Enterobacteriaceae (ESBL-PE) cause significant mortality and morbidity globally.  ESBL-PE cause a range of infections including uncomplicated UTIs, life- threatening bacteraemia, URTIs, gastroentritis, & colonising wound infections.  Mortality of patients with ESBL +ve sepsis is significantly higher than those with ESBL -ve sepsis – up to 30% of GNB-caused sepsis is fatal.  Are implicated in large scale outbreaks in hospital or community settings.  Cause localised or institutionalised outbreaks.  Infections caused by ESBL-PE are associated with rising healthcare cost.  Decreased productivity as a consequence of prolonged hospitalisation.  ESBL-PE are associated with increasing episodes of clinical treatment failure.

10  ESBL producing organisms have important therapeutic and clinical ramifications for patients from whom they are isolated.  ESBL-PE pose significant public health risks.  ESBL-PE pose serious infection control challenges.  ESBL production in Enterobacteriaceae has been a consequence of widespread use of broad spectrum antibiotics in hospital settings.  Increasing prevalence is reported in isolates recovered from community- based patients.  ESBLs are transferrable via conjugative plasmids thus dissemination of resistance genes among bacterial populations can occur and spread in larger geographic regions.  Treatment of ESBL-PE involves a combination of antibiotics, some of which have undesirable side effects including nephrotoxicity.

11  Risk factors for infections with ESBL-PE in healthcare- or community-acquired infections include; ◦ Previous use of antibiotics including broad spectrum antibiotics e.g 3GC cephalosporins; ◦ Recent or prolonged hospital admissions including admissions to ICU; ◦ Recurrent UTIs; ◦ Empiric antibiotic therapy ◦ Increased age; female gender; institutionalised residential care e.g. nursing homes; ◦ Intravenous therapy; ◦ International travels to areas of established endemicity e.g India subcontinent, the Middle East and Africa; ◦ Immunosuppressive chemotherapy; ◦ Invasive procedures- indwelling urinary catheters; central venous catheter, and ◦ Underlying comorbidities such as chronic renal insufficiencies, haemodialysis, liver disease, diabetes mellitus, malignancy, hypertension, heart disease, neutropenia, and HIV infection

12  ESBLs were first reported in Germany in 1983.  This followed introduction of broad spectrum 3G cephalosporins into clinical use.  ESBLs have been reported in all parts of the world – except Antarctica.  ESBLs are derivatives of classic β-lactamases eg SHV-2 is derived from SHV-1.  ESBLs are occasioned by single mutations in progenitor (parent) enzymes ◦ A mutation of few amino acids.  ESBLs exhibit fundamental changes in substrate spectra, substrate profile, reactions to inhibitors & isoelectric point – important distinguishing factors.  Over 200 ESBLs are characterised & classified – there is still no consensus on exact figure.  Β-lactamases have been variously classified over time.  Two commonly used classification schemes are; ◦ Ambler molecular classification system ◦ Bush-Jacoby-Medeiros functional classification system.

13  The Ambler molecular system classifies β-lactamases on the basis of protein homology (amino acid similarities); ◦ 4 major classes (A, B, C & D).  The Bush-Jacoby-Medeiros functional system classifies β- lactamases, on the basis of functional similarities/substrate and profile inhibitor profile; ◦ 4 main groups (1, 2, 3 & 4).  ESBLs are derived from group 2be β-lactamases; ◦ the `e’ of 2be denotes the extended-spectrum capability of the newly derived enzyme.  ESBLs are quite diverse.  Clinically important ESBLs are derived from 3 major types of classic beta-lactamases; TEM-, SHV-, & CTX-M-type β- lactamases. ◦ Temoniera – a Greek patient from whom this ESBL type was first isolated. ◦ SHV - Sulfhydryl Variable. ◦ CTX-M - Cefotaxime – Munich (first isolated in Munich)


15  Snapshot of major ESBLs – SHV -, TEM- & CTX-M-types including rare and peculiar ESBLs

16 Enzyme familyFunctional group or subgroup No. of enzymesRepresentative enzymes CMY1, 1e50CMY-1 to CMY-50 TEM2b, 2be, 2br, 2ber172 2b12TEM-1, TEM-2, TEM-13 2be79TEM-3, TEM-10, TEM-26 2br36 TEM-30 (IRT-2), TEM-31 (IRT-1), TEM- 163 2ber9TEM-50 (CMT-1), TEM-158 (CMT-9) SHV2b, 2be, 2br127 2b30SHV-1, SHV-11, SHV-89 2be37SHV-2, SHV-3, SHV-115 2br5SHV-10, SHV-72 CTX-M2be90CTX-M-1, CTX-M-44 (Toho-1) to CTX- M-92 PER2be5PER-1 to PER-5 VEB GES 2be 2f 7 15 VEB-1 to VEB-7 GES-2 to GES-7 (IBC-1) to GES-15 KPC2f9KPC-2 to KPC-10 SME2f3SME-1, SME-2, SME-3 OXA2d, 2de, 2df158 2d5OXA-1, OXA-2, OXA-10 2de 2df 9 48 OXA-11, OXA-14, OXA-15 OXA-23 (ARI-1), OXA-51, OXA-58 IMP3a26IMP-1 to IMP-26 VIM3a23VIM-1 to VIM-23 IND3a8IND-1, IND-2, IND-2a, IND-3 to IND-7 [i] [i] Enzyme families classified on the basis of amino acid structures (G. Jacoby and K. Bush, [ii] [ii] The sum of the subgroups in each family does not always equal to overall number of enzymes in each family due to withdrawn or non-classification of some enzymes.

17  Stats of ESBL epidemiology are profoundly varied – all parts of the world have different rates of prevalence.  In general terms; ◦ TEM-type ESBLs are predominantly reported in the United States, ◦ SHV-type ESBLs are most frequently isolated in Western Europe. ◦ CTX-M-type ESBLs have been detected in Australia, Latin America, Eastern Europe, and in specific countries such as Japan, Spain, & Kenya.  Global epidemiology captures in major surveillance studies; ◦ AGAR (Australia) ◦ SENTRY (US, Canada & Latin America) ◦ SMART ( Global - US, SE Asia) ◦ EARSS (European countries)

18 CountryStudy name or period K. pneumoniaeE. coli Number of isolates Percentage of ESBL positive Number of isolatesPercentage of ESBL positive CanadaSENTRY 1997- 1999 3864.912034.2 US and CanadaSENTRY 19981924.2-- USASENTRY 1997- 1999 20177.949663.3 USASENTRY 1997409447714.7 Latin AmericaSENTRY 1997- 2000 25543.911425.4 Latin AmericaSENTRY 1997- 2000 1274023310.0 Latin AmericaSENTRY 1997- 2000 66447.312396.7 Latin AmericaSENTRY 1997- 1999 89745.420268.5 EuropeSENTRY 1997- 1999 94622.638225.3 Italy199994620.0 46041.2 SpainEARSS 2001--19621.55 France1996-2000612111.4-- GermanyPEG 20012688.26190.8 Netherlands1997196<1571<1 Turkey19974348.85301.1 Western Pacific area SENTRY 1997- 1999 56024.611047.9 Asian Pacific area SENTRY 1998- 1999 67825.2133710.1 China19995595142723.6 Taiwan200012411.317711.9 Hong Kong19984721370211

19  Use of both genotypic and phenotypic techniques.  Phenotypic testing – a 2 steps process; ◦ Screening; screening process aims to exclude potential ESBL-producing isolates by testing for resistance or reduced susceptibility to 3GC cephalosporins.  Screening using cefotaxime, cefpodoxime, ceftazidime, and aztreonam discs.  multiple 3GC agents reliably improves sensitivity by offering wider ESBL substrate base. ◦ Confirmation; second step tests for synergy between 3GC cephalosporins & clavulanates (synergy between β-lactams and β-lactams-clavulanate combinations) – also known as DDST (double disc synergy test).  A disc zone diameter difference of ≥5 mm between a cephalosporin and its respective cephalosporin- clavulanate is taken as a phenotypic confirmation of ESBL production.  e.g an ESBL-producer tested against ceftazidime produces these resistance zones: ceftazidime zone = 16; ceftazidime-clavulanic acid zone = 21)  Automated (Vitek 2 systems) MBD ◦ Automated microbroth dilution - growth at or above screening concentrations (breakpoint) may indicate production of ESBL (that is, for E. coli and K. pneumoniae, MIC ≥ 2 μg/mL for ceftriaxone, ceftazidime, aztreonam, or cefpodoxime).  E-test, microScan panels and other discs-based methods are also used.

20 Can you tell a plate depicting ESBL positive in the Figure above?

21 SettingESBL positiveESBL negativeTotal Hospital30259289 community75402477 Total105661766 Frequencies at assigned age categories 0-20 years old21-40 years old41-60 years old≥61 years old 11261553

22 Comparison of percentage resistance of ESBL-producing isolates recovered from patients in hospital (HP) and community (CP) settings

23  What should be done to curb increasing threats pose by ESBL- mediated antibiotic resistance; ◦ Robust antibiotic stewardship – appropriate use of antibiotics ◦ Effective infection control measures in hospitals – effective preventive measures to curb transmission;  Contact precautions,  Hand hygiene,  Disinfections of inanimate objects, surfaces, medical devices in healthcare facilities ◦ Public education – antibiotic resistance awareness campaign. ◦ Controlling use of antibiotics in food chains – control & regulation of antibiotic use in agriculture. ◦ Immunization – preventative & indirect ◦ Development of newer, potent antibiotics against emerging multidrug resistant bacteria. ◦ Timely detection, and reporting of ESBL producing bacteria by medical laboratories. ◦ Instituting infection control measures in institutionalised care settings – eg nursing homes. ◦ Active screening for multi-drug resistant Enterobacteriaceae. ◦ Classifying ESBL-PE as notifiable infections???

24  Therapeutic options are very limited.  Treatment usually involves a combination of drugs.  These are usually the expensive, last line of antibiotics;  Carbapenems (e.g meropenem, ertapenem)  Fosfomycin.  β-lactam/β-lactam-inhibitor combination drugs (e.g Amoxicillin-clavulanate, piperacillin-tazobactam…etc) – supporting evidence from clinical studies is, however, controversial.  Limitation of therapeutic drugs is also compounded by other factors such as ; ◦ Site of infection, ◦ Severity of infection, ◦ Renal or liver functions of a patient, ◦ Age, ◦ Pregnancy or lactation status, ◦ Other medications the patient may be taking.

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