Extended Spectrum Beta Lactamases

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Extended-spectrum beta-lactamases (ESBLs) were first reported in 1983 (Knothe H, Shah P, Krcmery V, Antal M, Mitsuhashi S 1983) while plasmid-mediated AmpC beta-lactamases were reported in 1988 (Bauernfeind A, Chong Y, Schweighart S 1989). However, George A. Jacoby 2009 were stated that AmpC ß-lactamase of E.coli was the first bacterial enzyme that destroyed penicillin reported in 1940 even it was yet named. Normally ESBLs are mutant of plasmid-mediated ß-lactamases which are derived from older broad-spectrum ß-lactamases (ie. TEM-1, TEM-2, SHV-1), which have an extended substrate profile that permits hydrolysis of all cephalosporins, penicillins, and aztreonam. These enzymes are most commonly produced by Klebsiellaspp and Escherichia coli but may also occur in other gram-negative bacteria, including Enterobacter, Salmonella, Proteus,andCitrobacterspp., Morganella morganii, Serratia marcescens, Shigella dysenteriae, Pseudomonas aeruginosa, Burkholderia cepacia, andCapnocytophaga ochracea (Goussard S, Courvalin P 1999) Plasmid-mediated AmpC ß-lactamases have arisen through the transfer of chromosomal genes for the inducible AmpC ß-lactamase onto plasmids and it was the first enzyme that destroy penicillin ( George A. Jacoby 2009). In addition, our understanding of these enzymes is hampered by the lack of information. This is a consequence of most laboratories not attempting to detect ESBLs or plasmid mediated AmpCs. More clinical data and therapeutic outcome studies are needed. Currently, some investigators recommend that resistance to third-generation cephalosporins and aztreonam should be reported irrespective of the MIC if a plasmid-mediated AmpC β-lactamase is detected( Pai, H. et al. 2004) However, the scientists were argued that the emergence of resistance to ß-lactam antibiotics was begun even before the first ß-lactam, penicillin was developed. The first ß-lactamase was identified in Escherichia coli prior to the release of penicillin for use in medical practice (Patricia and Bradford October 2001). Furthermore, the Swedish investigators began a systematic study of the genetics of penicillin resistance in E. coli in 1965. Mutations with stepwise-enhanced resistance were termed ampAand ampB(George A. Jacoby January 2009) the age of penicillin saw the rapid emergence of resistance in Staphylococcus aureusdue to a plasmid-encoded penicillinase. This ß-lactamase quickly spread to most clinical isolates of S. aureus as well as other species of staphylococci (Patricia and Bradford OCT 2009).

The development of antibiotics via penicillin for use in medical practice remains one of the most significant advances in modern medicine (Rice 2008).Therefore; antibiotics have saved countless lives and continue to be a mainstay of therapy for bacterial infections. In addition to this, clinical success of the first ß-lactam, penicillin G benzylpenicillin prompted the search for development of additional derivatives. This search gave rise to the ß-lactam antibiotics in clinical use today (penicillins, narrow- and extended-spectrum cephalosporins, monobactams, and carbapenems) as Sarah and Robert mentioned. These classes of antibiotics are commonest structural feature that have been the highly reactive part of the Blactam ring (Drawz and Robert A. Bonomo 2010).

Section 2

Literature Review

Generally, Harada and his colleagues 2008 were states that “the production of extended-spectrum ß-lactamase (ESBL) is one of the most important resistance mechanisms that hamper the antimicrobial treatment of infections caused by Enterobacteriaceae. ESBLs are classified into several groups according to their amino-acid sequence homology”. In my opinion I would agree with this point that it is an important mechanism but mainly in KlebsiellasppandEscherichia coli rather than other Enterobacteriaceae.

In term of calcification ß-Lactamases are commonly classified according to two general schemes: the Ambler molecular classification scheme and the Bush-Jacoby-Medieros functional classification system (Bush, Jacoby, and. Medeiros. 1995). However, this came after the fist Jack and Richmond in 1973 and major recognition by Bush scheme and updated in 1995.

In genetic classification via Ambler molecular classification scheme class A has a molecular mass of approximately 29,000 Da which characterized by an active-site serine specially hydrolysis of penicillins. In contrast with group B that has an ezymes called metallo B-lactamases(MBLs) which hydrolyze ampicillin and some cephalosporins include carbapenems ( Patricia A. Bradford 2001).

In addition, Molecular structure classifications which fortunately not involved in our project were first proposed by Ambler et al, in 1980 when only four amino acid sequences of ß-lactamases were known. At that time a single class of serine enzyme was designated, the class A b-lactamases that included the Staphylococcus aureusPC1 penicillinase, in contrast to the class B metallo-b-lactamase from Bacillus cereus. The class C cephalosporinases were described by Jaurin and Grundstrom in (1981), and class D oxacillin-hydrolyzing enzymes were segregated from the other serine ß-lactamases in (1981).

Extended-spectrum ß-lactamases are developing group of ß-lactamases that have ability to hydrolyze third-generation cephalosporins and aztreonam. These enzymes were derived from genes for TEM-1, TEM-2, or SHV-1 by mutations which alert the amino acid configuration around the active site of these ß-lactamases (David L. Paterson and Robert A. Bonomo October 2005). This information's were not sufficient as Bush-Jacoby-Medeiros 1995 were states that the molecular structure classification does not include whether these enzymes are affecting ß-lactamases inhibitors or no. The instability of MIC of treatment for E.coli orK.peumonia or other Enterobacteriaceae since 1980s affecting this molecular classification.

Emergence in serious community-onset infections organisms that produce CTX-M enzymes have become the most prevalent type of ESBLs described during the past 5 years, most of them specifically from European and South American countries.( Canton and Coque 2006). The CTX-M ß- lactamases, now exceeding 50 different types which can be divided into five groups based on their amino acid identities: CTX-M1, CTX-M2, CTX-M8, CTX-M9, and CTX-M25 (Bonnet 2004) Furthermore, organisms producing specific CTX-M enzymes have been isolated from different countries. (Canton, Coque2006). In addition, the main problems of CTX-M enzymes is that they are not limited to nosocomial infection caused by K.peumonia sp, their potential spread is beyond the hospital environment (Laurent Poirel ICAAC 2010).

One of the main reasons for preferring other classification scheme (Bush, Jacoby) is instability of the gene expression that responsible in ESBL producing organisms. Nevertheless, with easily attainable sequence data, sequences of all important b-lactamases may become available, and an inclusive phylogenetic tree can be constructed correlating the relationships among the molecular and functional classes.

On the other hand, the phenotypic classification via Bush-Jacoby-Medeiros classification scheme groups B-lactamases according to functional similarities (substrate and inhibitor profile) into four main groups and multiple subgroups in this system. The classification scheme was much more immediate relevance to the physician or microbiologist in a diagnostic laboratory because it considers ß-lactamase inhibitors and ß-lactam substrates to be clinically relevant according to Bush-Jacoby-Medeiros 1995. Alternatively, the classification of ß-lactamases on the basis of function began when cephalosporin's ß- lactamases with high hydrolysis rates for cephalosporins, were differentiated from penicillinases, enzymes with good penicillin-hydrolyzing activity. This functional classification schemes was preferred and accepted among ß-lactamase researchers. This true especially when we follow the functional classification schemes history starting by the classification of Sawai et al.1968, describing penicillinases and cephalosporinases by using the response to antisera as an additional discriminator, Richmond and Sykes scheme in1973 that included all of the ß-lactamases from gram-negative bacteria described at that time, classifying the enzymes into five major groups on the basis of substrate profile. the extension of the Richmond and Sykes scheme by Sykes and Matthew in 1976, emphasizing the plasmid-mediated ß-lactamases that could be differentiated by isoelectric focusing, the scheme proposed by Mitsuhashi and Inoue 1981 in which the category‘‘cefuroxime-hydrolyzing ß-lactamase'' was added to the ‘‘penicillinase and cephalosporinase''classification and the groupings proposed by Bush et al in 1989 that included enzymes from all bacterial sources and that was the first scheme to try to correlate substrate and inhibitory properties with molecular structure.

In addition, the importance of this classification leads Bush-Jacoby and their colleagues to continue for further studies and researches. Consequence, they were expand and updated this classification (Bush et al 2010). The table below shows the last updated by Bush and their colleagues:

TABLE 1. Classification schemes for bacterial ß-lactamases, expanded from Bush et al. (16)

Bush-Jacoby Bush-Jacoby Molecular class Distinctive Inhibited by Defining Representative

characteristic(s) enzyme(s)

Group Medeiros (subclass) substrate(s) CA or TZB EDTA

(2009) group (1995)

1 1 C Cephalosporins NO NO Hydoloysis cephalosporin E.coli AmpC, P99

More than penicillin ACT-1,CMY-2,FOX-1

1e NIb C Cephalosporins NO NO Hydrolysis CAZ + oxyamino GC1,CMY-37


2a 2a A Penicillins YES NO Hydrolysis penicillin PC1

More than cephalosporin

2b 2b A Penicillin,early YES NO Similar than above TEM-1,TEM-2

Cephalosporins SHV-1

2be 2be A extended YES NO increase hydrolysis Oxyamino B-lactum

Spectrum Cephalo TEM-3 SHV-2CTX-M-15


2br 2br A Penicillins NO NO resistant to B-lactam inhibitors TEM-30,SHV-10

2ber NI A Extended-Cephalos- NO NO resistant to B-lactam inhibitors TEM-50

Porins & mombactum + Oxyamino b-lactum

2c 2c A carbenicillin YES NO increase to hydrolysis Crba P PSE-1,CARB

2ce NI A carbenicillin+ cefepime YES NO increase to hydrolysis Crba P+cefepem

2d 2d D Cloxacllin VARIABLE NO increase hydrolysis Cloxa or oxacillin OXA-1-10

2de NI D Extended-spectrum VARIABLE NO hydrolysis oxacillin+cloxa + OXA-11-15

Cephalosporin oxyamino B-lactam

2df NI D Carbapenems Variable No hydrolysis Carbapenems+Oxyamino B-l OXA-23

2e 2e A Extended-spectrum yes No Hydrolysis cephalosporin + inhibited CepA

+ Cephalosporins by clavulanic acid but not aztronam

2f 2f A Carbapenems Variable No Hydrolysis carbapenem +oxyamino B-l kcp-2

3a 3 B(B1) Carbapenems Hydrolysis broad-spectrum IMP-1,VIM-1

B(B3) No Yes + crbapenams but not monobatum CcrA, IND-1

3b 3 B(B2) Carbapenems Preferential hydrolysis L1,CAU-1,GOB-1,FEZ-1

NI 4 Unknown No Yes carbapenems CphA, Sfh-1

aCA, clavulanic acid; TZB, tazobactam.

bNI, not included.

(MINIREVIEW, Updated Functional Classification of ß-Lactamases Mar. 2010)

(Karen Bush1* and George A. Jacoby2, American Society for Microbiology)

As Jacoby Munoz-Price 2005 were states that “the common mechanism of resistance among Escherichia coli, Klebsiellapneumoniae, and other Enterobacteriaceae is through the production of ß-lactamases, which depends on the enzyme that inactivate certain ß-lactam antibiotics”. Not only Jacoby recognized this, the strains resistant to broad-spectrum cephalosporins have been increasingly recognized by Burwen, Banerjee, Gaynes (1994) and Itokazu, Quinn, et al (1996). However, the real mechanism was not stated due to lack of information in this field previously. Recently, Sarah M. Drawz and Robert A. Bonomo 2010 were explained that to enable bacteria to overcome B-lactam antibiotics there were four mechanisms. Theses mechanisms are:

1) Production of ß-lactamase enzymes which commen and important mechanism, 2) Changes in the active site of PBPs can lower the affinity of ß-lactam antibiotics. This will increase resistance to these antibiotics such as those seen in PBP2x of Streptococcus pneumonia. and Staphylococcus spp. 3) Decreased expression of outer membrane proteins(OMPs).

4) Exporting a wide range of substrates from the periplasm to the surrounding environment which called Efflux pumps.

Therefore the important that is the risk factors for infections by ESBL producing organisms must be identified to develop an effective strategies to limit these infections. Thus, epidemiology of ESBLs producing organisms can be discussed here.

The prevalence of extended-spectrum ß-lactamases worldwide defer in term of global epidemiology. For example, Daivd L Paterson and Robert .A. Bnomo 2005 were mentioned that in the north of France, the ESBL producing Klebsiella pneumoniae incidence was decreased from 19.7% in 1996 to 7.9% in 2000. This is an example from Europe while other countries are deferent in their percentage. One of the lager study that include from more than 100 European intensive care unitsperformed shows that the prevalence of Klebsiella pneumoniae was ranged from 3% in Sweden to 34% in Portugal. With reference to Daivd L Paterson study in France, it could be argued that implemented a good policy and procedure of protection can minimize the dissemination of ESBLs producing organisms.

In north of America, the assessment of prevalence of ESBL producing organisms according to moland and colleagues in 24 medical centers was 75%. This is not indicate an overall percent in the US even there were some studies which have been done in deferent area in the US. However in my opinion the prevalence needs to be estimated clearly (Daivd L Paterson and Robert .A. Bnomo 2005).

At South and Central of America via Brazil, Colombia and Venezuela 30to 60% of Klebsiella pneumonia was isolated from intensive care units. It is appeared that estimated percentage in all South and Central of America countries yet performed.

In Asia, Klebsiella pneumonia and E.col were isolated in China 30% for Klebsiella pneumonia and 24.5% for E.coli and 5 to 8% of E.coli in Korea, Japan Malaysia and Singapore (Daivd L Paterson and Robert .A. Bnomo 2005).

In the Middle East and Africa a number of outbreak were reported via in Nigeria and Kenya. The outbreaks were reported without any documents that indicated they were due to ESBLs producing organisms. It is apparent that in the poorest countries in Africa there was no interest in detecting ESBLs producing organisms. However, in the Kingdom of Saudi Arabia there was study that done by Kader and Kumar found that the prevalence of ESBL in Saudi Arabian for gram-negative isolates was more than 20% higher than those reported in some Indian studies (Kader and Kumar 2004). I disagree with this study because there was big deference in term of population between K.S.A and India.

The detection of ESBL-producing organisms in laboratories was an important requirement for appropriate management of patients, infection prevention and control efforts, as well as for tracking these organisms in surveillance systems. However, the procedure of detection of organisms producing ESBLs in clinical microbiology laboratories remains a worrisome issue and compliance ranges. A quality studies was undertaken by the World Health Organization and Centers for Disease Control (CDC) have raised concerns about the present capabilities of numerous clinical laboratories to detect organisms producing ESBLs. Stevenson, Samore, Barbera et al 2003 were indicted that a small percentage of clinical laboratories from rural hospitals in the USA routinely screened for ESBL producing organisms. He added in term of quality testing of laboratories outside the United States showed a small fraction of laboratories were able to specifically identify a highly resistant ESBL producing K. pneumoniae isolate. From over all published studies, it obvious, that some laboratories were interested in detecting ESBLs producing organisms.

There are several methods to detect ESBLs which depend on Kirby -Bauer disk diffusion test. The fists one called double -disk which was described by Jarlie et al. This method can be done by swabbing the organism onto a Mueller-Hinton agar plate, then amoxicillin-calculate is placed in the center of the plate and other disk of oxyyimino-Blactum are placed 30 mm from the amoxicillin-calculate disk. If there is an enhancement of the zone of inhibition this will be indicator of ESBL positive. In addition, the sensitivity of this method can be increased by reducing the distance between both disks from 30 to 20 mm as have been suggested. Similar method was performed by Jacoby and Han using other ß-lactam inhibitor sulbactam with increase in the enhancement zone at least 5 mm indicate that ESBL positive (Patricia a. Bradford 2001).

Other method called three-dimentional test which described by Thomas and Sanders. In this test the organism will be incubated onto Mueller-Hinton agar plate and slit is cut into the agar into which broth suspension of the test organism is introduced. Hence there is destruction in expected circular zone of inhibition for the antibiotic disk that place 3 mm from slit; this will indicted that ESBL positive. A good sensitivity was determined for this method but it was difficult to perform easily.

The National Committee for Clinical Laboratory Standards (NCCLS) or (CLSI) recommended for ESBL detection include screening by testing for growth in broth medium containing 1 µg/ml of one of ceftazidime, cefotaxime, ceftriaxone, aztreonam or cefpodoxime or by a disk diffusion equivalent.(NCCLS 2004) If growth occurs, then MICs are determined to either ceftazidime and/or cefotaxime with, and without, the addition of 4 µg/ml clavulanic acid. A decrease in MIC of less 3 twofold dilutions is considered positive.

Dr. Matthew Muller in 2004 stated the importance of identifying a specific ESBL which has implications with respect to transmission, control, and therapy of ESBL that produce organisms. He therefore argue that if an organism does not have multiple ß-lactamases a very simple tests can give a clue of a possible reason why of ß-lactam resistance. Due to the fact that they lack inducible AmpC genes and the best simple indicators for ESBL and/or AmpC plasmids are routine susceptibility data on E. coli and Klebsiella sp. He (Dr. muller) further explained why the presence of an ESBL is not always enough to raise the MIC of one or more of the third-generation cephalosporins into the resistant range for the detection of ESBL producers which is challenging. Most phenotypic strategies to detect ESBL screen for minimal elevations in MIC of less1 of the third generation cephalosporins or aztreonam

Recently, there was several commercial manufactures have been developed to detect ESBL either manual or automated via Vitek System. Interestingly, Rasheed et al(1997). reported that in strain of K. pneumonia specially SHV1 production it was lacking an outer membrane protein which caused false-positive ESBL. This is an indicator that ESBL determination may affected by some factors which yet discovered clearly.

Molecular detection of ESBL, the fist method of detection ESBLs was used DNA probes especially for TEM and SHV follow by developing other method until PCR -RFLP recently used. Here we are not going to give more details about molecular method because our project concentrated in the phenotypic method (Patricia a. Bradford 2001).

Many laboratories have difficulty in detecting ESBL-mediated resistance because of cost-cutting practices, while others are unaware of the relevant Clinical Laboratory Standard Institute (CLSI) or Health Protection Agency (HPA) guidelines. (Journal of Antimicrobial Chemotherapy 2005)

Section 3

Knowledge Gap

With reference to previous and current published studies in term of determination and classification of ESBLs producing organisms specially E.coli and K. pneumonia, obviously there is luck of information in this field generally in the kingdom of Saudi Arabia and specific in Riyadh region. Therefore, our project aimed to detect extended spectrum B-lactamase producing organisms looking specifically for E.coli and K. pneumonia in clinical isolated collection of 120 specimens from both inpatient and outpatient at Security Forces Hospital Riyadh using phenotypic method.

With luck of information genetically and phenotypically for those ESBL producing organism, thus it is essential that these studies must be performed and it might be repeated genetically for further study.

The question that arises here is the determination and classification of ESBL for both E.coli and K. pneumonia gives a good picture in which classes of ESBL are predominant or disseminated in this region or further study is needed?

Section 4

Project Justification

The significant of the study in this project can be formed in three ways: the first one is Epidemiology which can tell us how ESBLs locally disseminated and whether it can be increased or decreased. For instant, as mentioned above in north of France the incidence of ESBL that related to K. pneumonia was dramatically decreased from 19.7% in 1996 to 7.9% in 2000 (Patterson and Bonomo, 2005). Other example was study that done by Stobberingh, Arends et al 1999 inAmsterdam region at Academic Medical Center (AMC). This study previously in 1997 showed that the prevalence of ESBL among isolates of E. coli and Klebsiella spp. was <1%. In 2003, CTX-M ESBL genes were detected in the AMC for the first time, and the present study shows that the dissemination of four different CTX-M ESBLs in nine different species, including the non-fermenting gram-negative species. These two example shows decrease and increase of dissemination of ESBL producing organism locally. In September 2010 (50Years ICAAC) Dr Karen Bush and Dr Robert Bonomo were discussed the recent top ten classic papers in ß-lactamases, Dr Karen Bush was estimated the number of ß-lactamase Families Enzymes by 340 in 2000, while this number was increased up to 950 according to study that done by Bush & Jacoby, AAC 2010. They were showed some an important characteristics of some families which may play crucial role of understanding these ß-lactamases enzymes in order to perform good strategies of the treatment in the future.

The second one is Risk Factors, several studies showed that some risk factors are associated with development of colonization or infected by ESBL producing organisms. Since the initial description of ESBL production by K. pneumoniaeisolates in 1983, strains resistant to broad-spectrum cephalosporins have been increasingly recognized. It is obvious that the prolonged hospital stay, multiple antibiotics used and ICUs admissions are the major risk factors for colonization and infection with Multi- Drug Resistant Organisms including ESBL. Therefore, the main goal is to identify these risk factors in our area and to minimize the infection with ESBL through implement an appropriate policies and procedures of protection in order to reduce the dissemination of ESBL producing organisms. In addition, “the frequent surveillance studies from different regions across the world may help in updating the empirical antibiotics” (S. Shakil,a,_ S. Z. Ali et al 2010).and it can play an important role in the treatment of such multi- drug resistant organisms.

The third one is Sensitivity Pattern which can be explained with some details. Many studies show that those organisms are harbouring ESBL enzymes Multi- Drug Resistant organisms and can lead to series treatment challenges. Thus inappropriate of empirical treatment can caused increased in the risk of treatment failure or can cause death. Ariffin et al (2000). was found in his study that most of sepsis-related mortality was higher among those patients infected with Ceftazidim resistant K. pneumoniae.

Wong-Beringer et al. suggested that the choice of cephalosporin for empirical treatment may affect the outcome. This was supported by investigators conclusion that indicate in case of infected by ESBL producing organisms “failure of the treatment with cephalosporins might occur at higher rates with Ceftazidime than other third-generation cephalosporins”.

In term of treatment for infection with ESBL producing organisms there is some strategies of treatment using Carbapenems , Pipercillin- tazobactam, Fluoroquinilones, Amino glycoside and Cefepime.

Carbapenems.Wong-Beringer et al (2002) states that imipenem has higher success rate in clinically treatmen for those patient infected by ESBL producing organisms via E. coli and Klebsiellapneumoniae. The Carbapenems were used successfully to control outbreaks that caused by ESBL producing organisms and imipenem was preferred in these cases (Ruben Ramphal et al 2006).

Pipercillin- tazobactam. This antibiotic contains combination of semi synthetic penicillin and β-lactam inhibitors. From over all studies that evaluating Pipercillin- tazobactam the data suggested that Pipercillin- tazobactam was useful against some infection with ESBL producing organisms.

Aminoglycosids.These antibiotics showsome variable activities againstthose organisms producing ESBLs. amikacin is the most active one than other aminoglycosid groups according to certain studies (Ruben Ramphal et al 2006).

Fluoroquinilones.These groups have limited successful in the treatment of ESBL produing organisms but it was helpful in certain cases.

Cefepime. As known is firth-genration of cephalosporinwhich more stable comparing with third-generation of cehalosporins. Moreover, in some clinical reports demonstrated that use of cefepime was successful with ESBL producing organisms via Enterobacter sp. It is suggested that using cefepime reduce the prevalence presumptive ESBL producing organisms (Ruben Ramphal et al 2006).

In 2010, the phenotypic analysis of resistant according to the CLSI which was defined for ceftazidime and/or cefotaxime by MIC > 2 µg /ml as indicative of ESBL production will not be always consider as break point of sensitivity to third generation cephalosporin's for enterobacteriaceae.

Section 5


The presence of ESBL producing organisms is increasingly affecting the course and results of an infection which raises a lot questions as well as poses a challenge to infection management worldwide. The challenge it has posed and it has some negative effect to the community which can be pandemic if adequate steps are not taken to curb its spread. There ought to be a well define approach towards identification of ESBL. Isoelectric focusing (IEF) has been suggested by Dr.Pitout as a useful technique that can identify the presence of one or more β-lactamases.

Antibiotic resistance is an important issue affecting public health, and rapid detection in clinical laboratories is seeing as the most essential means for the prompt recognition of antimicrobial-resistant organisms. Furthermore, it is obvious that ESBL producing organisms have its effect mainly in the ceftazidime or cefotaxime. In addition, the empirical and directed treatments were an effective treatment strategies recently performed. However, the impact of ß-lactamase induction in the clinic is very hard to measure when testing or evaluating antibiotics in preclinical or clinical trials, ß-lactamase induction may bear importance as an eventual predictor of efficacy. It is essential for infection-control practitioners and clinicians to bring the appropriate clinical laboratory equipments to rapidly identify and characterize different types of resistant bacteria efficiently to minimize the spread of these bacteria and help to select more appropriate antibiotics for treatment. In addition, physicians must take care to prescribe appropriate antibiotics in order to minimize the rate of spread of drug resistant bacteria.