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 Introduction of ESBL

ESBL stands for Extended-spectrum β- lactamase. In modern medical practice, newer antimicrobials drugs have been used extensively resulting in the emergence and rapid dissemination of resistant bacterial strains. Since one of the mechanisms of bacterial resistance to a β-lactam antibiotic is the production of β- lactamase enzyme that breaks down the structural β- lactam ring of penicillin and its synthetic derivatives. The property of stability to many bacterial β- lactamase was increased with the later generations of cephalosporins. However, the persistent exposure of bacterial strains to a multitude of β- lactams has induced a dynamic and continuous production and mutation of lactamase in many bacteria, expanding their activity even against the third and fourth-generation cephalosporins. These new β- lactamases are called ESBLs which were first reported in Germany.

ESBL Detection Method

Screening Method

Antibiotics      Zone of Inhibition (ZOI)

Ceftazidime(30µg)  ≤ 22 mm

Cefotaxime (CTX ) ≤ 27 mm

Cefpodoxime (CPD) ≤ 17 mm

Confirmatory method

Put ceftazidime (30 µg) and combination of ceftazidime (30 µg) and clavulanic acid ( 10  µg) disks with a distance of 20 mm from disk to disk on Muller-Hinton agar as shown above picture.

After overnight incubation, ZOI of ceftazidime clavulanic acid (CAC) should be equal to and greater than 5 mm that of ceftazidime.

The above isolate is showing the ESBL producer.

Other methods of detection-

Double disk Synergy Test (DDST): 

The disk of third-generation cephalosporin was placed at a 15 mm distance from amoxicillin-clavulanic acid. Enhanced inhibition indicates ESBL.

Micro Dilution Test:  Growth in a broth containing 1 µg/ml third-generation cephalosporins indicates ESBL.

MIC Broth Dilution: 

MIC of third-generation cephalosporin alone or combined with CA. A decrease in the MIC of the combination of 3 twofold dilutions indicates ESBL.

E- Test (MIC ESBL Strip): 

Two-sided strip containing CAZ on one side and CAZ- CA on the other. The ratio of MIC of the combination to that of CAZ alone > 8 or the presence of a phantom zone (or both) indicates ESBL.

Automated Instruments (Vitek): Measures MICs and compares the growth of bacteria in presence of CAZ vs. CAZ-CA.

Molecular (DNA probes, PCR, RFLP): Targets specific nucleotide sequences to detect different variants of TEM and SHV genes.

Details of ESBL 

ESBLs are β- lactamases containing serine at the active site and belong to Ambler’s molecular class -A (majority of them). They hydrolyze extended-spectrum cephalosporins with an oxyimino side chain. These cephalosporins include Cefotaxime, ceftriaxone, and ceftazidime, as well as the oxyimino- monobactam aztreonam. ESBLs confer resistance to expanded spectrum, aztreonam, and related oxyimino β- lactams; but they are not specific to cephamycins (e.g. Cefoxitin and cefotetan) or carbapenems (e.g. Meropenem or imipenem). Typically they derive from genes for TEM 1, TEM 2, or SHV 1 by a mutation that alter the amino acid configuration around the active site of this β- lactamases. This extends the spectrum of β- lactam antibiotics susceptible to hydrolysis by these enzymes. They are also generally susceptible to β- lactamase inhibitors, such as clavulanate, sulbactam, and tazobactam, which consequently can be combined with a β-lactam substrate to test for the presence of this resistance mechanism. The majority of ESBLs contain a serine at the active site. These enzymes are most commonly produced by Klebsiella spp.  and E. coli but may also occur in other gram-negative bacteria, including Enterobacter, Salmonella, Proteus, Citrobacter, Morganella morganii, Serratia marcescens, Shigella dysenteriae, P. aeruginosa, Burkholderia cepacia, Capnocytophaga ochracea.

TEM β- lactamases (Class A): Although TEM type β- lactamase is most often found in E. coli and K. pneumoniae, they are also found in other species of gram-negative bacteria with increasing frequency. The amino acid substitutions responsible for the ESBL phenotype cluster around the active site of the enzyme and change its configuration, allowing access to oxyimino β – lactam substrates. Opening the active site to β lactam substrate also typically enhances the susceptibility of the enzyme to β- lactamase inhibitors such as clavulanic acid. Single amino acid substitution at positions 104,164, 238, and 240 produce the ESBL phenotype, but ESBL with the broadest spectrum usually have more than a single amino acid substitution. Based upon different combinations of changes, currently, 140 TEM-type enzymes have been described.

SHV β- lactamases (Class A): These have amino acid changes around the active site, most commonly at positions 238 or 238 and 240. More than 60 SHV varieties are known. They are the predominant ESBL type in Europe and the United States and are found worldwide.

CTX – M – β lactamases (Class A): These enzymes were named for their greater activity against Cefotaxime than other oxyimino β- lactam substrates (e.g. ceftazidime, ceftriaxone, or cefepime). More than 40 CTX M enzymes are currently known. Despite their name, a few are more active on ceftazidime than Cefotaxime. They have mainly been found in strains of Salmonella enterica serovar Typhimurium and E. coli, but have also been described in other species of Enterobacteriaceae and are the predominant ESBL type in parts of South America. CTX- M- 14, CTX- M -3, and CTX-M -2 are the most widespread.

OXA β- lactamases (Class D): The OXA- type β -lactamases confer resistance to ampicillin and cephalothin and are characterized by their high hydrolytic activity against oxacillin and Cloxacillin and the fact that they are poorly inhibited by clavulanic acid. Amino acid substitutions in OXA enzymes can also give the ESBL phenotype. While most ESBLs have been found in E. coli, K. pneumoniae, and other Enterobacteriaceae, the OXA- type ESBLs have been found mainly in P. aeruginosa. Some confer resistance predominantly to ceftazidime, but OXA -17 confer greater resistance to Cefotaxime and Cefepime than it does resistance to ceftazidime.

Other β- lactamases: Other plasmid-mediated ESBLs such as PER, VEB, GES, and IBC β- lactamases have been described but are uncommon and have been found mainly in P. aeruginosa and at a limited number of geographic sites.

Treatment option

ESBL producing isolates typically show greater than average resistance to other agents including aminoglycosides and fluoroquinolones. These relationships were illustrated in a review of 85 episodes of bacteremia due to ESBL production from 12 hospitals in seven countries. All isolates were susceptible to imipenem or meropenem, while 71% were resistant to gentamycin, 47% to piperacillin-tazobactam, and 20% to ciprofloxacin. When an oxyimino- β – lactam is used to treat severe infections caused by ESBL- producing K. pneumoniae, treatment failure is likely even if the organism tests susceptible to the antibiotic in vitro. In a review of 28 patients with ESBL – producing K. pneumoniae with reported susceptibility to cephalosporins, 15 failed to respond to cephalosporin therapy. A high degree of associated resistance to gentamycin, co-trimoxazole, and quinolones was found in ESBL producers. The majority of ESBL producers were detected among patients. Currently, carbapenems are generally regarded as the preferred agent for the treatment of infections due to ESBL- producing organisms. Carbapenems are resistant to ESBL- mediated hydrolysis and exhibit excellent in vitro activity against strains of Enterobacteriaceae expressing ESBLs.

Clinical outcome due to ESBL

ESBL producing bacteria are typically associated with MDR^. The antibacterial choice is often complicated by multi-drug resistance. Thus, infection due to ESBL producing bacteria can result in avoidable failure of treatment and increased cost in patients who have received inappropriate antibiotic treatment. Colonization and infection with these bacteria have also been associated with indiscriminate use of antibiotics, prolonged hospitalization, increasing numbers of immune-compromised patients, and medical progress resulting in increased use of invasive procedures and devices. Once an ESBL producing strain is detected the laboratory should report it as ‘resistant” to all penicillins, cephalosporins, and aztreonam, even if they test as susceptible. So, updated knowledge of the susceptibility pattern of bacteria is important for the proper selection and use of antimicrobial drugs and for the development of an appropriate prescribing policy. The first prevalence study of ESBL producing bacterial isolates in Nepal by Pokhrel et al in 2005 showed that > 20% isolates were positive for ESBL. In this context, it is quite necessary to present the current scenario of ESBL production in our setting. Problems in identification arise because ESBLs are heterogeneous. OXA- type ESBLs e.g. are poorly inhibited by clavulanate. Some ESBLs are best detected with ceftazidime and others with cefotaxime (such as most CTX- M enzymes). Consequently, susceptibility to several oxyimino β –lactams must be tested. In the combination disk method, E. coli ATCC 25922 is used as ESBL negative control, and K. pneumoniae ATCC 700603 is used as ESBL positive references strains (132). Because different ESBLs hydrolyze β – lactams at different rates, several different agents must be examined to examine their presence.

Worldwide pattern of ESBL

 The prevalence of ESBLs among clinical isolates varies from country to country and from institution to institution.

This difference may be due to geographical variations, local antibiotic prescribing habits, etc. Although the prevalence of ESBLs is not known, it is clearly increasing, and in many parts of the world, 10-40 % of strains of E. coli and K. pneumoniae express ESBLs.

In the United States, the occurrence of ESBL production in Enterobacteriaceae ranges from 0 to 25%, depending on the institution, with the national average being around 3%. In Korea and Indonesia, the distribution of ESBL in E. coli is 5% and 23.3% respectively which is higher when compared to North America or Europe, but similar to that of South America. In Japan, the percentage of β- lactam resistance due to ESBL production in E. coli and K. pneumoniae remains very low. In a recent study of 196 institutions across the country, < 0.1% of E. coli and 0.3% of K. pneumoniae strains possessed and ESBL. Elsewhere in Asia, the percentage of ESBL production in E. coli and K. pneumoniae varies like 8.5% in Taiwan93 and 12% in Hong Kong, The first prevalence study of ESBL producing bacterial isolates in Nepal by Pokhrel et al showed that > 20% of isolates were positive for ESBL. In this context, it is quite necessary to present the current scenario of ESBL production in our setting.

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