Sulbactam

Background & objectives: The widespread use β-lactam antibiotics has lead to the development of resistance to this group of antibiotics in bacterial pathogens due to β-lactamase production. Information on such pathogens is not available from eastern region of India. This study was undertaken to determine the AmpC β-lactamase production in pathogens isolated from hospitalized patients in Kolkata.

Methods: Non-repeat clincal isolates (284) from pus, urine, sputum and other clinical specimens of hospitalized patients were taken. Disk agar diffusion (DAD) and minimum inhibitory concentration (MIC) with different β-lactam antibiotics, and double disc synergy test (DDST) with clavulanic acid and sulbactam were done. Disk antagonism test (DAT) and three-dimensional extract test (TDET) were conducted for phenotypic confirmation of AmpC and inducible AmpC β-lactamase production. Nitrocefin spot test and microiodometric assay of β-lactamase were also performed.

Results: Twenty seven isolates were found to he resistant to cefoxitin, a α-methoxy-β-lactam. Of these, 19 were observed to be AmpC β-lactamase producers and 4 were inducible AmpC β-lactamase producers by DDST, DAT and TDET. Remaining 4 were non AmpC β-lactamase producers. Of the 23 AmpC β-lactamase producers, the distribution of different species was as follows: Escherichia coli 11 (47.8%), Pseudomonas aeruginosa 4 (17.3%) Klebsiella pneumoniae 3 (13%), and Klebsiella aeruginosa 1 (4.3%).

Interpretation & conclusion: Our finding showed 6.7 per cent AmpC β-lactamase and 1.4 per cent inducible AmpC β-lactamase producing clinical isolates from Kolkata. AmpC β-lactamase producing bacterial pathogens may cause a major therapeutic failure if not detected and reported in time.

Key words AmpC β-lactamase - cefoxilin - clavulanic acid - inducible AmpC β-lactamase - sulbactam - three-dimensional extract test (TDET)

A common mechanism of bacterial resistance to β-lactam antibiotics is the production of β-lactamase enzymes that cleave the structural β-lactum ring of these drugs. This is the predominant mechanism of β-lactam resistance in Gram-negative bacteria1,2. Over the last two decades many new β-lactams have been developed that were specifically designed to be resistant to hydrolytic actions of β-lactamase2. However, with this new class of drug that has been used to treat patients, new types of β-lactamases emerged. AmpC β-lactamase is one of these new types of β-lactamases.

AmpC β-lactamases are cephalosporinases, which belong to the molecular class C as classified by Ambler in 1980' and Group I under a classification scheme of Bush et al4. AmpC β-lactamases are more sensitive to inhibition by sulbactam than by clavulanate or tazobactam5. These are clinically significant as they may confer resistance to a wide variety of β-lactam drugs, including α-methoxy-β-lactams, narrow, expanded and broad-spectrum cephalosporins, aztreonam, a monobactam4 and most significantly β-lactam plus β-lactamase inhibitor combinations (viz., ampicillin-clavulanic acid, pipericillin-tazobactam, etc.).

In many species, β-lactamases are normally produced at very low levels but are induced to several hundred fold higher by the presence of β-lactams (viz., cefoxitin, cefotaxime, etc.) and certain β-lactam inhibitors (viz., clavulanic acid)6. Inducible AmpC β-lactamases are such examples. Amoxicillin-clavulanic acid combination is commonly used in controlling β-lactamase producing pathogens, as clavulanic acid acts as an inhibitor to many β-lactamases. But in case of inducible AmpC β-lactamases, this type of drug can cause more harm than help.

β-lactamase producing bacteria can cause serious therapeutic failure if not detected on time. Though the clinicians treat infections based on the results of antibiotic susceptibility tests available, the number of infections caused by AmpC β-lactamase producing organisms is on the rise and pose a threat to the patients due to treatment failure. A few groups have reported the occurrence of β-lactamases producing bacteria from northern and southern regions, but there are not much data available on these pathogens from Kolkata or anywhere in the eastern region. The present study was therefore undertaken to find out the presence of AmpC and inducible AmpC type of β-lactamases producing clinical isolates from hospitalized patients in Kolkata using standard methods presently available for their detection. It may be mentioned here that currently there is no clear consensus regarding guidelines for performing tests for the phenotypic screening or confirmatory tests for the isolates that harbour AmpC β-lactamases7,8.

Material & Methods

Bacterial isolates: A total of 284 non-repeat clinical isolates collected from patients admitted to various wards during February 2002 to April 2003 [90 (31.7%) from pus, 132 (46.5%) from urine, 57 (20.1 %) from sputum, 5 (1.8%) from others specimens such as burns, catheter, throat swab, ear discharge, gastric lavage fluid, and peritonial fluid] from five hospitals in Kolkata, namely Calcutta Medical College and Hospital (CMC), Nil Ratan Sarkar Medical College (NRS), Seth Sukhlal Karnani Memorial Hospital (SSKM), R.G. Kar Medical College (RGK) and School of Tropical Medicine (STM) were included in this study.

Media and chemicals: Ampicillin (A, Lyka Labs, India), amoxycillin (Am, Wyeth Lederle Ltd, India), amoxicillin/clavulanic acid (Am/CA, Ranbaxy Laboratories, India) aztreonam (Ao, Hi-media, Mumbai), cefactam (Cfs, Aurobindo Pharma Ltd., India), cefotaxime (Ce, Alkem Laboratories Ltd, India), cefpodoxime (Cep, Universal Medicare Pvt, India), ceftriazone (Ci, Wockhardt, India), ceftazidime (Ca, Glaxo, India), cefoxitin (Cn, Hi-media, Mumbai), cefpirome (Cpm, Alkem, India), cephalexin (CpI, Glaxo, India), ciprofloxacin (Cf, Ranbaxy, India), clavulanic acid (CA, Glaxo Smith Kline, UK), cotrimoxazole [Co, (sulphamethazole/ trimethoprim),Welcome, India], gentamicin (G, Nicolas Piramal, India), imipenem (IPM, Ranbaxy Laboratories, India), piperacillin/tazobactam (Pt, Himedia, Mumbai), tetracycline (T, Hoesct, India), were used in this study. Antibiotic solutions were prepared in sterile water. For disc agar diffusion (DAD), minimum inhibitory concentration (MIC) and double disk synergy test (DDST) Muller-Hinton broth (MHB, Hi-media, Mumbai) and agar agar (Qualigen Fine Chemicals, India) were used.

Disc agar diffusion method (DAD): The test bacterium, taken from an over-night culture (inoculated from a single colony) was freshly grown for 4 h and with this culture a bacterial lawn was prepared on MHA plate. Filter paper disks of 6 mm size were used to find out antibiotic susceptibility pattern against 10 antibiotics (concentration in µg) [A (10), Am (20). Ao (30). Cf (5). Cn (30). Co (sulphamelha/ole/ trimethoprim. 1.25/23.75). G (10). T (30). CpI (30) and IPM (1O)). four third generation cephalosporins (Ca (30), Ci (30). Ce (30), Cep (10)). one fourth generation cephalosporin [Cpm (30)). three β-lactam+β-lactamase inhibitor combination, viz., amoxicillin-clavulanic acid combination [Am/CA. (20/10)). pipericillin-tazobaclam combination [Pt. (100/10)] and cefapera/one-sulhactam [Cfs (75/30)) combination following Kirbs-Bauer method9. The disks were prepared according to manufacturer's instruction. Strains resistant tocefoxitin (zone diameter less than 18 mm) were suspected to be AmpC β-laclamase producers.

Minimum inhibitory concentration (MlC): The MIC of the antibiotics was determined by two-fold serial broth dilution method10.

Double disk synergy test (DDST): In DDST. synergy was determined between a disk of cefotaxime (30 µg) and a disk of cefotaxime plus C A (30µg + 10µg) which were placed at a distance of 20 mm apart on a lawn of culture of the suspected β-lactamase producing clinical isolates on MHA. Disks containing clavulanic acid were prepared by applying 10 µl of a 1000 µg/ml clavulanic acid stock solution to each disk. The test organism was considered to produce β-lactamase if the zone size around the cefotaxime plus clavulanic acid increased >5 mm in comparison to the third generation cephalosporin (Ce) disk alone. This increase occurred because the β-laclamases produced by the isolates were inactivated by clavulanic acid. The NCCLS guidelines do not have urn standard tests for isolates, that produce AmpC β-lactamase8. If the increase in zone size was

Disk antagonism test (DAT): The disk antagonism lest was used to detect the inducibility of β-lactamase. Disks of inducing agent cefoxitin (Cn) and cephalosporins (Cpm, Ca. Ci and Ce) were placed on the surface of the test bacterial lawn on MHA plates on a lawn of bacterial culture of the suspected inducible AinpC β-lactamase producers separated by 15 mm. The plates were examined after overnight incubation at 37°C12. If blunting of the cephalosporin disks adjacent to the cefoxitin disks occurred, the organisms were considered to produce inducible AmpC β-lactamase.

Three-dimensional extract test (TDET): 50 µl of a 0.5 McFarland bacterial suspension prepared from an overnight MHB was inoculated into 12 ml of MHB and the culture was grown for 4 h at 35°C. The cells were concentrated by centrifugation, and crude en/.yme preparations were made by sonicating the pellets at 8 µm (in Soniprep. UK) for 15 sec (two cycles) with IO sec cooling in between sonicalions; this was repealed four times. The surface of a MHA plate was inoculated with Escherichia coli strain ATCC 25922 (obtained from Central Drug Laboratory. Kolkata) as described in DAD. A 30 µg cefoxitin (Cn) disk was placed at the center of the inoculated agar. With a sterile scalpel blade, a slit beginning at 5 mm from the edge of the Cn disk was cut in the agar in an outward radial direction. By using a pipet, 25 to 30 µl of enzyme prepation was dispensed into the slits, beginning near the disk and moving outward. Slit overfill was avoided. The inoculated media were incubated overnight at 35°C. Enhanced growth of the surface organism at the point where the slit intersected the zone of inhibition due to Cn was considered a positive TDET result and was interpreted as evidence for the presence of AmpC β-lactamase7.

Nilrocefin spot test: The crude enzyme was prepared by centrifuging overnight culture of the organisms at 10.000 r.p.m at 4°C for 10 min. The pellets were then sonicated at 8 µm (in Soniprep, UK) for 15 sec (two cycles) with IO sec cooling in between sonications. The sonicated material was centrifuged again to obtain the enzyme; 10 µl of enzyme was incubated with 50 µl of 1.5 mM nitrocefin (Calbiochem. San Diego. USA; working solution prepared according to the manufacturer's instruction) in the well of a microtitre plate for 30 min at room temperature. The presence of β-lactamase was detected if colour of nitrocefin changed from yellow to reddish-orange13.

Microiodometric determination of β-lactamase activity: Microiodometric determination was done according to the method of Sykes and Nordstrom14 using benzylpenicillin and cefotaxime as substrates.

Induction of AmpC β-lactamase by clavulanic acid: For testing, induction of AmpC β-lactamase by clavulanic acid, 10 µg/ml of clavulanic acid was added to MHB in which the test bacteria were grown overnight at 37°C in a gyratory shaker. Enzyme was prepared from this overnight growth as described previously. The enzymes thus obtained were tested by nitrocefin spot test and microiodometric determination of β-lactamase activity.

Isoelectric focusing: Analytical isoelectric focusing was performed with some of the β-lactamase extracts, by the method of Mathew et α/13. The ampholine range used were pH 3.5-10. The samples were loaded on the gel and given a pre-run of 20 min at 15 mA at constant volts of 1 Watt and then run for 60 min at 50 mA on LKB Multiphor II isoelectric focusing apparatus (Pharmacia, Sweden), β-lactamases with known isoelectric point (pI) were focused as control: 15 µl of TEM 1 (pI 5.4) obtained from E. coli J53 RI (Dr Reddy's Laboratory, Hyderabad) and 10 µl of SHV-18 (pi 7.8) obtained from Klebsiella pneumoniae ATCC 700603 (Dr Reddy's Laboratory, Hyderabad) were used. Gels were stained with 10^sup -4^ M nitrocephin. The pi of the enzyme was indicated by a reddish-orange band on the gel bed at a specific position.

Results

Of the 284 non-repeat clinical isolates tested by DAD, 27 (9.5%) were resistant to cefoxitin which was used as a primary selection criterion of AmpC β-lactamases. MIC as well as DDST done on these 27 isolates with Ce, Ce/CA and Cfs showed that 19(12 from urine, 3 from pus and 4 from sputum and throat swabs) were resistant to inhibition by clavulanic acid but were inhibited by sulbactam (Table I, Fig.1) and tazobactam. Four isolates showed decreased zone of inhibition with clavulanic acid suggesting production of β-lactamases induced by clavulanic acid (Table II). Of the 27 isolates, the remaining four were nonproducers of AmpC β-lactamase. All the 23 isolates, which produced AmpC β-lactamase, exhibited high level of resistance to the antibiotics tested by DAD. Seventeen of the AmpC β-lactamase producers were resistant to ciprofloxacin. Ceftazidime showed sensitivity in 9 (39%) of the 23 isolates tested positive for AmpC β-lactamase production. Cefpodoxime resistance was seen in all 23 isolates.

AmpC β-lactamase production was confirmed by performing TDET. All the suspected 19 isolates were found to be positive for TDET, i.e., there was growth along the slit within the zone of inhibition of cefoxitin (Fig. 2). Inducibility of the β-lactamases was further recognized by DAT, which demonstrated blunting of specific cephalosporin (cefotaxime) disks adjacent to the cefoxitin disks (Fig. 3).

AmpC β-lactamase production (including inducible AmpC β-lactamases) was seen in 23 (8.1%) isolates. AmpC β-lactamases were produced by E. coli 11 (47.8%), P. aeruginosa 4 (17.3%), K. pneumoniae 3 (13%) and K. aeruginosa 1 (4.3%). Of the inducible AmpC β-lactamase producing isolates, 3 were isolated from urine samples (K. pneumoniae CMC-40, Proteus vulgaris CMC-90 and P. aeruginosa NRS-3) and 1 from burn patient (P. aeruginosa NRS-226) (Tables I, II).

All AmpC β-lactamase producers were found to be positive by nitrocefin spot test, β-lactamase activity from all the 23 isolates were also tested using benzylpenicillin and cefotaxime as substrates following the microiodometric assay. The specific activity of β-lactamase enzyme varied from one organism to another but all were positive for βlactamase production. Four isolates were found to be producing inducible AmpC β-lactamase which was also confirmed enzymatically. Addition of clavulanic acid in the growth medium induced the production of inducible AmpC β-lactamase in case of both the substrates, benzylpenicillin and cefotaxime, from 1.1 fold to 16 fold, confirming that clavulanic acid is an inducer of this enzyme in vivo (Table III).

Isoelectric focusing showed that both TEM-I (pi 5.4) and SHV-18 (pi 7.8) type of β-lactamase enzymes were the predominant types of enzyme present in the AmpC β-lactamases (Figs 4, 5). In case of some samples including the reference strain E. coli J53RI, a band is seen at the point of application (Figs 4, 5); this may be due to aggregation of proteins which did not migrate under the experimental conditions applied.

Discussion

The efficacy of β-lactam group of antibiotics was reduced due to the production of β-lactamases by the resistant bacterial strains. Therefore, search for their inhibitors was initiated to protect the antibiotic activity in vivo against β-lactam resistant pathogens. Clavulanic acid, a naturally occurring β-lactam, had been the first such inhibitor, which is produced by Streptomyces clavuligerus15. Subsequently, a few more viz., sulbactam, a penicillanic acid sulphone16, tazobactam, etc., were found.

Plasmid mediated AmpC β-lactamase from K. pneumoniae isolates was first reported in 1989 from Seoul, South Korea17. Within 1998, nineteen types of plasmid mediated AmpC β-lactamases were reported from Algeria, France, Germany, Greece, India, Pakistan, Taiwan, Turkey, United Kingdom and United States'8. The prevalence of AmpC β-lactamase enzyme was 2 per cent in E. coli and 17.1 per cent in K. pneumoniae in China19. Recently, AmpC-type β-lactamase producing K. pneumoniae were also reported from the Republic of Korea20. Plasmid mediated inducible AmpC β-lactamases are still rare. DHA-1 type of inducible AmpC β-lactamases were first reported from Saudi Arabia in 199821 and later on from Taiwan in 200212. Six of the 51 isolates of Enterobacteriaceae were found to be inducible AmpC β-lactamase producers from Korea22. In Richmond, Virginia, USA, 2.6 per cent of K. pneumoniae were found to be AmpC β-lactamase producers23. Plasmid encoded AmpC type β-lactamases were found in 8.5 per cent of the K. pneumoniae, 6.9 per cent of the K, oxytoca and 4.0 per cent of the E. coli collected from 25 US capital states and the district of Columbia24.

In 2003, 20.7 per cent AmpC enzyme producers were found among Gram-negative bacteria in Guru Tegh Bahadur Hospital, Delhi25. In the same year Subha et al found AmpC β-lactamase production in 24.1 per cent of Klebsiella spp. and 37.5 per cent of E. coli in Chennai26. Shahid et al found 20 per cent of P. aeruginosa producing AmpC β-lactamase in Aligarh27, and in Karnataka, 3.3 per cent of E. coli, 2.2 per cent of K. pneumoniae, 5 per cent of C. freundii, and 5.5 percent off. aeregenes (all urinary isolates) were found to harbour AmpC enzymes28.

The phenotypic data generated in this study indicated that in Kolkata hospitals (6.7%) of the isolates were phenotypically confirmed to he AmpC β-lactamase producers and 1.4 per cent to be inducihlc AmpC β-lactamase producers which were less than that reported from Delhi25 Chennai26 and Aligarh27 but more than that was found in Karnataka28.

It has been reported that Amp C β-lactamases had pI values like that of TEM-1, SHV-11 and DHA-1 type of β-lactamases, which are pH 5.4, 7.6. and 7.8. respectively12. In our study, we also found that many of the AmpC β-lactamases are having pi values in the range oi pH 5.4 and pH 7.8.

This is perhaps the first report of AmpC β-lactamase and inducible AmpC β-lactamase producing bacteria from Kolkata. India. Clavulanic acid if used as an inhibitor of β-lactamase in the AmpC β-lactamase producing bacteria, can cause therapeutic failure. If the type of β-lactamase produced by the pathogen could be detected along with the antibiograni before administering the β-lactam drug to the patient, therapeutic failure might be avoided. These changes in the bacterial population represent evolutionary upgrades, which provide them a greater potential to resist β-lactam antibiotics and cause formidable therapeutic and diagnostic challenges.

Acknowledgment

The authors thank Dr P.C. Banerjec. Deputy Director. Indian Institute of Chemical Biology. Kolkata for help in experimental design. Dr Dhrubojyoti Chatiopadhyay. Dean of Science, Department of Biochemistry. UCS&T. Ballygunj. Kolkata. for providing facilities for isoclcciric focusing. Dr Magadi Sila Ram. Dr Reddy's Laboratory for providing A', pneumoniae ATCC 700603 and E. coli J53RI. Dr M.K. Mazumdar. Ex-Director. Central Drug Laboratory. Kolkata. for providing Escherichia coli ATCC 25922. We thank Ranbaxy Pharmaceutical for supplying the imipenem disks and Glaxo-Smilh Klinc for providing clavulanic acid.

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Suranjana Arora & Manjusri Bal

Section of Microbiology. Department of Physiology. University of Calcutta. University College of Science & Technology, Kolkata, India

Received June 4, 2004

Reprint requests: Dr Manjusri BaI, section of Microbiology, Department of Physiology, University of Calcutta University College of Science & Technology, 92, A.RC. Road, Kolkata 700009, India

e-mail: manjusrb@vsnl.net

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