Novel β-lactamases in the Clinical Isolates of Enterobacter spp. and Klebsiella pneumoniae in Ramadi General Hospital: A Pharmacodynamics Study

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1 Novel βlactamases in the Clinical Isolates of Enteobacte spp. and Klebsiella pneumoniae in amadi Geneal Hospital: A Phamacodynamics tudy Majid A. K. Lafi Bc, Mc, PhD* iwa Mustafa Mohammed Bc, Mc** Hamdi Abdul Jalil AlHadithi BVM, Mc, PhD*** Abstact Backgound: Enteobacte spp. and Klebsiella pneumoniae ae among the most common gamnegative pathogens associated with hospital infections, they have a distinctive ability to develop antimicobial esistance duing theapy due lagely to thei unusual βlactamases, AmpC and EBLs. Aim of tudy: To investigate the occuence of novel βlactamases (AmpC and EBLs) in clinical isolates of Enteobacte spp. and K pneumoniae based on Phamacodynamics appoaches. Mateials and Methods: A total of 165 samples collected fom ALamadi Geneal Hospital epesented by 15 bun swabs, 95 wound swabs and 55 uine samples fom in and outpatients fom Octobe 2003 to Apil 2004, by API 20 E system and motility test 13 isolates have been identified to be Enteobacte spp. and 33 isolates to be K pneumoniae fom a total 150 isolates in addition to fou isolates of Enteobacte spp. identified fom 10 isolates taken fom a pevious study on Klebsiella spp. The susceptibility of the isolates to βlactam antibiotics wee detemined by the standad disk diffusion method as descibed in the guidelines of the National Committee fo Clinical Laboatoy standads (NCCLs). Appopiate tests wee pefomed fo detecting AmpC βlactamase, confiming this detection, inducibility of AmpC βlactamase, and EBL poduction. esults: The intepetive eading of the antibiogam fo Enteobacte spp. 1. Classical AmpC inducible enzyme: 2/17(11.8%) 2. EBLs enzyme: 10/17(58.8%) 2. The numbe of deepessed o patially deepessed mutants wee 7/17 (41.2%) that epesenting 7/14 (50%) of E. cloacae. While, fo K pneumoniae infes the following: Classical low HV1 βlactamase 2/33 (6.1%), All Enteobacte spp. isolates wee esistant to cefoxitin but 15/17 wee positive in the AmpC βlactamase inhibition and the 3DM tests. Out of the 33 isolates of K pneumoniae one isolate was esistant to cefoxitin and showed positive esult in the AmpC βlactamase inhibition and the 3DM tests. even isolates of Enteobacte spp. showed blunting effect in the zone diamete of used βlactams by cefoxitin o imipenem. Accoding to the double disk synegy test fo detecting the EBLs enzyme, 8/17 (27.1%) of ou Enteobacte isolates and 19/33 (57.6%) of K pneumoniae positive in giving phantom phenomenon. Conclusion: It is concluded that the high occuence of the novel βlactamases in the clinical isolates of Enteobacte spp. and K pneumoniae infes a poo contol of the use of antibiotics and infection in this hospital. Key wods: Enteobacte spp, Klebsiella pneumoniae, AmpC, EBLs Intoduction nteobacte spp. ae among the most common E gamnegative pathogens associated with hospital infections 1, they have a distinctive ability to develop boad spectum antimicobial esistance duing theapy, owing lagely to thei AmpC β lactamases 2. The expession of AmpC enzyme is typically inducible in Enteobacte spp. and facilitates the emegence unde antibiotic pessue of highly esistant stably deepessed mutants which poduce lage quantity of the enzyme that can hydolyse the expanded spectum cephalospoins and penicillin but emains susceptible to cabapenems 3,4,5. The poduction of extendedspectum β lactamases (EBLs) has been documented in Enteobacte spp. Which was fist epoted in 1983 in K. pneumoniae and E. coli 1. Typically, EBLs ae mutant, plasmidmediated βlactamases deived fom olde, boadspectum βlactamases (e.g., TEM1, TEM2, HV1), which have an extended substate pofile that pemits hydolysis of all cephalospoins, penicillins, and azteonam except cephamycins such as cefoxitin. These enzymes ae most commonly poduced by Klebsiella spp. and Escheichia coli but may also occu in othe gamnegative bacteia, including Enteobacte 6,7. Genes fo AmpC βlactamase ae notably absent in K. pneumoniae 8 and found on plasmids that tansfe to K. pneumoniae which ae believed to be oiginated fom the chomosome of othe Enteobacteiaceae 9,8. It is of theapeutic inteest fo a clinical laboatoy to distinguish between these βlactamases as esistance makes due to inceasing numbe of epots of AmpC o EBLs poducing oganisms fo which the MICs of expandedspectum βlactams ae low and which ae associated with theapeutic failues 10,9. The aim of this study was to investigate the occuence of novel βlactamases (AmpC β lactamase and EBLs) in clinical isolates of Enteobacte spp. and K pneumoniae based on Phamacodynamics appoaches. Because of scace local epots on such clinically impotant esistance makes and to pomote local awaeness on such issues, it is felt wothwhile publishing the finding of this study seveal yeas afte pefoming it. Mateials and Methods 124 Iaqi J. Comm. Med., Ap (2)

2 Novel βlactamases in the clinical isolates of Enteobacte spp. & Klebsiella pneumoniae in amadi Geneal Hospital Majid Lafi et.al. A total of 165 samples collected fom AL amadi Geneal Hospital epesented by 15 bun swabs, 95 wound swabs and 55 uine samples fom in and outpatients fom Octobe 2003 to Apil 2004, by API 20 E system and motility test 13 isolates have been identified to be Enteobacte spp. and 33 isolates to be K pneumoniae fom a total 150 isolates in addition to fou isolates of Enteobacte spp. identified fom 10 isolates taken fom anothe study on Klebsiella spp. The susceptibility of the isolates to βlactam antibiotics wee detemined by the standad disk diffusion method as descibed in the guidelines of the national committee fo clinical laboatoy standads 11. The susceptibility to cefoxitin disk (30 μg) was used as a pimay sceening fo detection of AmpC βlactamase and the isolate with zone inhibition diamete < 18 mm wee selected 9 and then tested fo AmpC βlactamase inhibition by cloxacillin 8. In ou study cloxacillin was used as a phamacodynamic tool (indicato) fo the detection of the poduction of AmpC βlactamase in isolates using disk diffusion technique as a synegy effect with cefoxitin as an AmpC induce (30 μg fo each). The theedimensional method (3DM) was used as confimatoy test fo the detection of AmpC β lactamase in the positive isolates fom the sceening tests as descibed by Coudon et al 9 with the exception that bacteial lysis was not pefomed, i.e. whole cell suspension was used. The test fo inducibility of AmpC βlactamase by cefoxitin, and imipenem was pefomed by the disk appoximation method 12. EBL poduction was detemined using double disk synegy test (DDT) descibed by Livemoe and Bown 13. Intepetive eading of the antibiogam data was pefomed to investigate the unusual esistance mechanisms fom esistance phenotype 14. esults: The esults of API 20 E system wee as the following: out of 150 isolates 13 (8.7%) have been identified to be Enteobacte spp. (positive onithine decaboylase tests and being motile) distibuted as (7,4 and2) and 33(22%) isolates to be K pneumoniae (negative onithine decaboylase test and non motile) distibuted as (17, 5, 11) fom wound, bun and uine samples espectively. The occuence of Enteobacte spp in uine samples was low compaed with that of wound and bun swabs and lowes than that of K. pneumoniae, but it was high in bun swabs (26.7%) and slightly less than that of K. pneumoniae isolates (33.3%). The 13 isolates of Enteobacte spp. including 11 isolates of E. cloacae and 2 isolates of E. gegoviae. Fou isolates of Enteobacte spp. obtained fom 10 isolates taken fom a pevious study on Klebsiella spp. 15 ; one of them was identified to be E. aeogenes while the emaining 3 isolates wee found to be E. cloacae. In the pimay sceening test fo AmpC β lactamase all Enteobacte spp. isolates wee esistant to cefoxitin but 15/17 wee positive in the AmpC βlactamase inhibition test (figue 1A) and the thee dimensional method (3DM) (figue 1B). Out of the 33 isolates of K pneumoniae one isolate was esistant to cefoxitin and showed positive esult in the AmpC βlactamase inhibition test and the thee dimensional method. The intepetive eading of the antibiogam data descibed in tables (1) fo Enteobacte spp. and (2) fo K. pneumoniae. even isolates of Enteobacte spp. showed blunting effect (antagonism effect) in the zone diamete of used βlactam by cefoxitin o imipenem induced enzyme (figue 1C). Accoding to the double disk synegy test fo detecting the EBLs enzyme 8/17(27.1%) of ou Enteobacte isolates and 19/33 (57.6%) of K. pneumoniae positive in giving phantom phenomenon (figue 1D). A B C D OX T CO OX CN T CAZ AUG Figue.1. A. showing the extension of the inhibition zone aound the cefoxitin disk () towads the cloxacillin disk (OX) indicating the inhibition of AmpC enzyme. B. enhanced gowth of the suface oganism, E. coli ATCC at the point whee the slit, containing Enteobacte spp., intesected the zone of inhibition is consideed as positive 3DM indicating the pesence of AmpC enzyme; CN: contol negative isolate, T: AmpC enzyme poducing isolates. C. detection of an inducible AmpC enzyme in E. cloacae, the disk with lage zone is (CAZ) with 30 µg and the disk with vey small zone is cefoxitin 30 µg. Note the blunting of the inhibition zones by the cefoxitininduced enzyme. D. DDT. K. pneumoniae shows the phantom phenomenon between Coamoxiclav (Augmentin, AUG) with ceftiaxone (CO). 125 Iaqi J. Comm. Med., Ap (2)

3 Novel βlactamases in the clinical isolates of Enteobacte spp. & Klebsiella pneumoniae in amadi Geneal Hospital Majid Lafi et.al. Table (1): phenotypes: intepetation of mechanisms of antibiogam: βlactam vesus Enteobacte spp. Isolate no. AMP AMX/CLAV PIP CEF CXM CAZ CTX CO FEP ATM IMP EBLs test Induction test Infeed β lactamase type E. cloacae B 3 E. cloacae U 10 E. cloacae B 4 E. cloacae B 5 E. cloacae W 6 E. cloacae B 7 E. cloacae W 8 E. aeogenes B 15 E. cloacae W 9 E. cloacae W 11 E. cloacae W 1 E. cloacae W 2 E. cloacae B 13 E. cloacae B 14 E. cloacae B 12 E. gegoviae W 16 E. gegoviae U 17 / Typical classical AmpC inducible Enzyme AmpC enzyme and EBL Ceftazidimase 17 AmpC enzyme and EBLBoad Hypepoduction of AmpC deepessed enzyme Patially deepessed enzyme 18 EBL? 6 The mechanism of esistance was intepeted accoding to the intepetative eading 6, 14 and othewise stated. B: bun, W: wound, U: uine Table (2): phenotypes: intepetation of mechanisms of antibiogam: βlactams vesus K. pneumoniae AMP AMX/CLAV PIP CEF CXM CAZ CTX CO FEP ATM IPM EBLs test Infeed β lactamase type ve 2/2 Classical low HV1 / ve 8/8 Penicillinasehigh level ve 6/10 ve 4/10 ve 10/10 ve 1/1 The mechanism of esistance was intepeted accoding to the intepetative eading 6, 14. EBLBoad EBLCeftazidimase Acquied AmpC enzyme & EBLs Discussion: pecies of Enteobacte ae becoming inceasingly impotant nosocomial pathogens although they wee much less commonly encounteed than Escheichia coli and Klebsiella stains 1. Most nosocomial Enteobacte infections appea to aise endogenously fom a peviously colonized site in the involved patient 3. The esults of this study demonstated the occuence of Enteobacte spp. was high in bun swabs (26.7%) and slightly less than that of K. pneumoniae isolates (33.3%) due to the selective envionment with bun patients like loss of fluids and immunoglobulins with low tissue viability and low dug delivey so the high use of antibiotic theapy which suppesses the othe nomal floa and povides an excellent oppotunity fo colonization by 126 Iaqi J. Comm. Med., Ap (2)

4 Novel βlactamases in the clinical isolates of Enteobacte spp. & Klebsiella pneumoniae in amadi Geneal Hospital Majid Lafi et.al. Enteobacte spp. 3, but in the case of uine sample whee the dug is concentated in the site of infection and can eliminate the oganism 14. The phenotypic tests fo AmpC detection ae not well defined 6 and the NCCLs documents do not indicate the sceening and confimatoy tests that should be used fo the detection of the AmpC β lactamase in K. pneumoniae 9. In ou study all Enteobacte spp. showed esistance to cefoxitin indicating pimaily the pesence of AmpC βlactamase. Of the cephamycins, cefoxitin was used as sceening agent fo AmpC β lactamase because this antibiotic is stable against the activity of multiple βlactamase like TEM1, 2, HV1 and EBLs but hydolysed by AmpC enzyme 16. Theefoe, the esistance to cefoxitin may be due to the pesence of AmpC enzyme o eduction in oute membane pemeability 6. Out of 17 of ou Enteobacte isolates, 15 showed extension in the zone of inhibition aound the disk of cefoxitin towads the cloxacillin disk (30 μg) and these isolates yielded positive esults in the 3DM indicating the pesence of AmpC βlactamase. Enteobacte gegoviae known to poduce vey low noninducible level of AmpC βlactamase 4 and that ou two isolates showed negative esults in the cloxacillin inhibition test and 3DM and the esistance to cefoxitin may be due to the eduction in oute membane pemeability 9. Out of 33 isolates of K. pneumoniae, one isolate (3%) was esistant to cefoxitin and gave positive esults in the cloxacillin inhibition test and 3DM, thus, infeing the pesence of acquied plasmid mediated AmpC βlactamase, and not eduction in the oute membane pemeability, to be esponsible fo the esistance to cefoxitin 19. Accoding to the DDT in ou study 8/17 (47.1%) of Enteobacte spp. and 19/33 (57.6%) of K. pneumoniae wee found to be EBLs poduces. In the study of Canton et al (2003) in pain showed that (35%) of Enteobacte spp. wee EBLs poduces by DDT. ALJumaily 15 epoted that (52%) of clinical Klebsiella isolates wee EBLs poduces and AlKubaisy 20 epoted that 19/35 (54.28 %) of Klebsiella isolates collected fom amadi Geneal Hospital wee EBLs poduces in DDT. All EBLs poducing isolates should be epoted as esistant to all pencillins, cephalospoins and azteonam because these isolates have a damatic inoculum size effect with these agents 6. The detection of EBLs in AmpC poducing oganisms ae moe difficult 21 that high level expession of AmpC may pevent ecognition of an EBLs and in addition the AmpC enzyme may be induced by clavulanate and then attack the indicato cephalospoin, thus masking any synegy aising fom inhibition of an EBLs and poducing falsenegative esult 13. The high level of AmpC expession has minimal effect on the activity of cefepime making this dug a moe eliable detecting agent fo EBLs in the pesence of an AmpC βlactamase 6, theefoe, the K. pneumoniae isolate that was esistant to cefoxitin, gave the phantom phenomenon with cefepime only. Upon the induction test, seven isolates of ou Enteobacte spp. showed a positive esult by flattening (blunting) of the antibiotic inhibition zone on the side facing the cefoxitin o imipenem disks while the emainde isolates consideed to be deepessed mutant and/o EBLs poducing isolates 22. Enteobacte spp. and Klebsiella spp. ae impotant clinical pathogens that fequently exhibit esistance to thidgeneation cephalospoins. In Enteobacte spp. stains, esistance is usually due to deepession of the AmpC locus, wheeas plasmidencoded EBLs ae pimaily esponsible fo esistance in Klebsiella spp. 22. The esult of sensitivity testing showed that Enteobacte spp. wee nonsensitive to a wide ange of βlactam antibiotics used but they showed good sensitivity to imipenem (100%). Ampicillin and amoxicillin, fist and second geneation cephalospins ae stong AmpC βlactamase induces in wild type of Enteobacte spp. and apidly inactivated by these βlactamases while thid geneation cephalospins, extended spectum penicillins and azteonam ae weak induces and the esistance is expessed in stably deepessed bacteia (mutant hypepoduces of βlactamase) 23. Hence, the high esistance in ou study isolates may infe the pesence of AmpC deepessed mutants. The poblem is that AmpC inducible oganisms may be isolated fom a patient and found to be susceptible to thid geneation cephalospoins when, howeve, these dugs ae used clinically, thee is intheapy selection of deepessed esistant mutants, with highe possibility of theapeutic failue. This isk is as high as 20% of Enteobacte bacteaemia, it is theefoe ecommended that all Enteobacte spp. should be epoted esistant to all thid geneation cephalospins, except whee these isolates ae fom uinay tact whee vey high cephalopoins concentations can be attained 13. While in K. pneumoniae we showed also high esistance to the extended spectum cephalospoins and azteonam but (97%) of them showed sensitivity to cefoxitin and (100%) to imipenem, that may efe to the pesence of EBLs poducing isolates, EBLs enzymes can hydolyze all βlactam antibiotics but emain non active against cephamycins and ae most pevalent in Klebsiellae especially in intensive cae and othe specialized units 13. Imipenem ae stong AmpC βlactamase induce but emain vey stable to the action of these enzymes 1. The appaent absence of esistance mechanism to imipenem may be attibuted to being effectively esistant to all the common plasmid encoded class A, C and D enzymes including extended spectum TEM and HV deivatives, and AmpC types Iaqi J. Comm. Med., Ap (2)

5 Novel βlactamases in the clinical isolates of Enteobacte spp. & Klebsiella pneumoniae in amadi Geneal Hospital Majid Lafi et.al. Theefoe, this level of sensitivity indicates the absence of class B enzymes (Metallo βlactamase) and cabapenamase which encoded chomosomally and belong to class A βlactamase 16,14. AL Kubaisy 20 epoted that 7/25 (28%) of K. pneumoniae isolates wee esistant to imipenem and 1/25 (4%) wee Metallo βlactamase poduce that showed esistance to ceftazidime, cefotaxime, ceftiaxone and imipenem despite the fact that, to the date of conducting the study, imipenem has not been used clinically in ALamadi Geneal Hospital, and thee is no cossesistance confeed by the usage of cephalospoins to imipenem. In addition the detection of metallo β lactamase needed a test fo inhibition by EDTA 8 and this vey high occuence of esistance to imipenem is difficult to econcile consideing that the available body of liteatue states that esistance to imipenem is unusual to encounte in Enteobacteicae (except with Poteus spp.). It has been advised that when such esult is encounteed the species identification and antibiogam data should be echecked 14. An appoach has been intoduced to ead and utilize antibiogam data by (Intepetative eading) that aims to analyze the esistance patten. Not just the esults fo individual antibiotics, and so to pedict and ecognize the unusual and infeing esistance mechanisms fom esistance phenotypes to achieve bette theapeutic outcome 14. The intepetive eading of ou data in table (1) showed that 2/17 (11.8%) of ou Enteobacte spp. wee designated as classical inducible AmpC β lactamase. the poblem with inducible AmpC poducing oganism that may be isolated fom a patient and found to be susceptible to the thid geneation cephalospoins, when, howeve these dugs ae used clinically, thee is selection of deepessed esistant mutants with contingent clinical failue especially in seveelyill patients, theefoe, should be epoted as esistant to all thid geneation cephalospoins 14. Out of 17 isolates, 10 (58.8%) wee EBLs poducing (60%) of them wee EBLs ceftazidimase and (40%) poducing EBLs boad enzyme. The two isolates of E. gegoviae failed to poduce positive EBLs confimatoy test; it has been suggested that hypepoduction of TEM and HV βlactamase o eduction in oute membane pemeability in oganisms with EBLs may cause falsenegative phenotypic confimatoy test esults 6. AmpC deepessed enzyme poducing isolates wee found to epesent 4/17(23%) which showed high esistance to all βlactams except imipenem and consideed to be as deepessed mutants 24. One out of 17 (5%) isolate gave positive esult in induction test with some βlactams infeing to be patially deepessed mutant 18 and showed negative esult in EBLs detection. It is suggested that the numbe of deepessed o patailly deepessed mutants wee 7/17 including two isolates showed positive EBLs with cefepime only and the seven isolates wee E cloacae 7/14 (50%). Pai et al 18 in his study in Koea epoted that (50%) of Enteobacte spp. wee fully o patially deepessed mutant and (6.9%) wee classical inducible βlactamase (wildtype). The Intepetative eading of K. pneumoniae antibiogam patten (table2) showed the high pecentage of EBLs poducing isolates 23/33 (69.7%) and only one of them (3%) poduced both EBLs and acquied AmpC enzyme that showed phantom phenomenon with cefepime only and esistant to cefoxitin 14. K. pneumoniae acquied plasmids encoding AmpC enzyme that have escaped fom othe Enteobacteiaceae 25, 8. ALJumaily 15 epoted that (8%) of Klebsiella spp. in his study poduced plasmid mediated AmpC enzyme, and Coudon et al 9 epoted that (1.1%) of K. pneumoniae in his study poduced this enzyme. The high pecentage stated by the fome 15 was pobably due to misidentification of Enteobacte spp as Klebsiella spp. and this was evident afte passing ten of his study isolates to be echecked duing the couse of this study, fou of the ten isolates wee found to be Enteobacte spp. Futhe, it has been suggested that plasmidmediated AmpC enzymes pesent the geatest theat clinically by seiously limiting theapeutic choices, even moe so than EBLs 26. It is concluded that the high occuence of the novel βlactamases in the clinical isolates of Enteobacte spp. and K pneumoniae infes a poo contol of the use of antibiotics and infection in this hospital. Futhe, although clinically the usual occuence of Enteobacte spp. is less than that of K pneumoniae, in cetain clinical settings like bun, the ability of the fome to expess unusual multiesistance mechanisms favouing selection and emegence of esistant clones makes them moe difficult to contol. Ou clinical laboatoies should catch up with new bacteial developments; othewise, new pathogens will spead, esulting in inceasing teatment outcome poblems and costs fo patients and institutions. efeences 1. Fase L, Anett M, inave CP. Enteobacte Infections: emedicine Infectious Diseases. Updated: Jan 7, medescape.com. Enteed 29/1/ Jacoby GA. AmpC βlactamases. Clin Micobiol ev 2009; 22: andes WE, andes CC. Enteobacte spp.: pathogen poised to flouish at the tun of the senty. Clin Micobiol ev 1997; 10: Pitout JDD, Moland E, Thomson K, andes CC, Fitzsimmons. βlactamases and detection of βlactam esistance in Enteobacte spp. Antimicob Agents Chemothe 1997; 41: chwabe MJ, Gaham C, and BE, Gold H, Cameli Y. 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