Daniel Garang Kuir. BBioMedSci, USQ M App Sci (MedSci), RMIT 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 AmpCproducers. K. pneumoniae & E. coli are major producers of ESBLs in this group of gram negative bacteria. 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 thirdgeneration cephalosporins, most penicillins, and betalactam/beta-lactam-inhibitor (BL/BLI) combinations. 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 βlactamases (e.g TEM, SHV or CTX-M). 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-, & thirdgeneration 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 chromosomallyencoded AmpC β-lactamases – thus presence of ESBLs may be masked by AmpC. 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. Source: Rosário NA, Grumach AS. Allergy to beta-lactams in paediatrics: a practical approach. J Pediatr (Rio J). 2006;82(5 Suppl):S181-8. Source: Partridge, S. (2014). Movement of resistance genes in hospitals. Microbiology Australia. ESBL-producing Enterobacteriaceae (ESBL-PE) cause significant mortality and morbidity globally. ESBL-PE cause a range of infections including uncomplicated UTIs, lifethreatening 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. 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 communitybased 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. 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 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. 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 βlactamases, 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 βlactamases. ◦ Temoniera – a Greek patient from whom this ESBL type was first isolated. ◦ SHV - Sulfhydryl Variable. ◦ CTX-M - Cefotaxime – Munich (first isolated in Munich) Snapshot of major ESBLs – SHV -, TEM- & CTX-M-types including rare and peculiar ESBLs Enzyme family Functional group or subgroup CMY 1, 1e TEM 2b, 2be, 2br, 2ber SHV No. of enzymes 50 CMY-1 to CMY-50 172 2b 2be 2br 12 79 36 2ber 9 2b, 2be, 2br Representative enzymes TEM-1, TEM-2, TEM-13 TEM-3, TEM-10, TEM-26 TEM-30 (IRT-2), TEM-31 (IRT-1), TEM163 TEM-50 (CMT-1), TEM-158 (CMT-9) 127 2b 2be 2br 30 37 5 SHV-1, SHV-11, SHV-89 SHV-2, SHV-3, SHV-115 SHV-10, SHV-72 CTX-M 2be 90 PER VEB GES 2be 2be 2f 5 7 15 CTX-M-1, CTX-M-44 (Toho-1) to CTXM-92 PER-1 to PER-5 VEB-1 to VEB-7 GES-2 to GES-7 (IBC-1) to GES-15 KPC SME 2f 2f OXA 2d, 2de, 2df 9 3 KPC-2 to KPC-10 SME-1, SME-2, SME-3 158 2d 2de 2df 5 9 48 OXA-1, OXA-2, OXA-10 OXA-11, OXA-14, OXA-15 OXA-23 (ARI-1), OXA-51, OXA-58 IMP 3a 26 IMP-1 to IMP-26 VIM IND 3a 3a 23 8 VIM-1 to VIM-23 IND-1, IND-2, IND-2a, IND-3 to IND-7 Enzyme families classified on the basis of amino acid structures (G. Jacoby and K. Bush, http://www.lahey.org/studies/). [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. [i] 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) Country Study name or period Italy SENTRY 19971999 SENTRY 1998 SENTRY 19971999 SENTRY 1997 SENTRY 19972000 SENTRY 19972000 SENTRY 19972000 SENTRY 19971999 SENTRY 19971999 1999 Spain France Germany Netherlands Turkey Western Pacific area Asian Pacific area China Taiwan Hong Kong EARSS 2001 1996-2000 PEG 2001 1997 1997 SENTRY 19971999 SENTRY 19981999 1999 2000 1998 Canada US and Canada USA USA Latin America Latin America Latin America Latin America Europe K. pneumoniae Number of isolates 386 E. coli Percentage positive of ESBL Number of isolates Percentage positive of 4.9 1203 4.2 192 2017 4.2 7.9 4966 3.3 409 255 44 43.9 771 114 4.7 25.4 127 40 233 10.0 664 47.3 1239 6.7 897 45.4 2026 8.5 946 22.6 3822 5.3 946 20.0 4604 1.2 6121 268 196 43 560 11.4 8.2 <1 48.8 24.6 1962 619 571 530 1104 1.55 0.8 <1 1.1 7.9 678 25.2 1337 10.1 559 124 472 51 11.3 13 427 177 702 23.6 11.9 11 ESBL 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. A disc zone diameter difference of ≥5 mm between a cephalosporin and its respective cephalosporinclavulanate 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) ◦ 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). 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. Can you tell a plate depicting ESBL positive in the Figure above? Setting ESBL positive ESBL negative Total Hospital 30 259 289 community 75 402 477 105 661 766 Total Frequencies at assigned age categories 0-20 years old 21-40 years old 41-60 years old ≥61 years old 11 26 15 53 100% % Resistant_HP 90% % Resistant_CP 80% Percentage resistance 70% 60% 50% 40% 30% 20% 10% 0% AMP AMC TIM TZP FOX CRO MEM GM Major classes of antibiotics CIP FT SXT Comparison of percentage resistance of ESBL-producing isolates recovered from patients in hospital (HP) and community (CP) settings What should be done to curb increasing threats pose by ESBLmediated 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??? 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.