Detection of inhibitor resistant beta-lactamases in a tertiary care centre of Manipur, India: a report of three cases.
Although the organisms possessing these enzymes are resistant to combinations of beta-lactams and beta-lactamase inhibitors, they remain susceptible to narrow spectrum (Cephalothin) and extended spectrum cephalosporins (Ceftazidime, Ceftriaxone), carbapenems and monobactams (8). Most of the cases showed resistance to amoxicillin/clavulanate and ampicillin/sulbactam as when compared to piperacillin/tazobactam. (8)
We report three isolates of Enterobacteriaceae that resist the activity of [beta]-lactamase inhibitors.
CASE REPORTS: Case 1: A 34 years old lady from an urban area of Manipur, India presented to the outpatient department of medicine, JNIMS, Imphal on 7th December, 2012 with complaints of intermittent lower pain abdomen, frequency of micturition, dysuria and nausea for about 1week. On examination, body temperature was 38[degrees]C, hypogastrium was tender and other systems were normal. She was advised urine microscopy with culture and sensitivity, and ultrasonography of whole abdomen. USG showed normal study.
Case 2: A 67 years old man, farmer, smoker from a rural area of Manipur was admitted to male medicine ward, JNIMS, Imphal with fever, productive cough of pink-tinged sputum, exertional dyspnea and malaise for 3 weeks. There was no significant past or family history. On examination, he was underweight, body temperature 39[degrees]C, pulse 120 beats/min, BP-134/90 mmHg and respiratory rate 38 breaths/min. Auscultation of the chest revealed diffusely reduced breath sounds, rhonchi and faint inspiratory crackles in the lower right lung. There was no clubbing. Other systems were normal. Routine blood, blood sugar (Random), sputum microscopy and culture sensitivity, chest X-ray were sent. Empirical treatment of ceftriaxone and azithromycin was started. Blood test revealed leucocytosis (17,000/[mm.sup.3]), blood sugar of 90mg% and consolidation in right lower lobe of lungs on chest X-ray.
Case 3: A 28 year old lady from a rural area attended the outpatient department of medicine, JNIMS, Imphal on 12th August, 2013 with fever, chills, abdominal discomfort, urinary frequency and urgency, and dysuria for 5 days. She had similar episode one month back for which she took medicines from a pharmacy without consulting doctor. Examination showed body temperature of 39[degrees]C, tachycardia, mild pallor, mild tenderness of hypogastrium. Routine blood, urine microscopy and culture sensitivity, and USG of lower abdomen were advised. Her Hb was 9gm%, WBC-8000/[mm.sup.3] and normal USG.
Microbiological Studies of the Cases: Grossly, both the urine specimens were turbid with pH of 5.5 and 7 in case 1 and 3 respectively. Microscopically, plenty of pus cells were seen. The sputum was purulent with pinkish tinge and direct gram stain revealed gram negative bacilli with plenty of pus cells and few epithelial cells.
Identification and antibiotic sensitivity pattern of the causative bacteria from the clinical samples were performed by the Vitek 2 system (Bio Me'rieux, Marcy l'Etoile, France) following presence of pure culture of isolated colonies of the microorganisms on blood agar and Mac Conkey's agar. (9) Following the manufacturer's instructions, they were inoculated onto the identification and the AST-N280 cards, on which different concentrations of eighteen antibiotics were assayed. This system also features an Advanced Expert System (AES) that interprets the antibiotic resistance patterns, validates the results and reports the resistance phenotype, and has proved useful in calculating MIC values.
E. coli was isolated in both the urine samples and Klebsiella pneumoniae in the sputum. However, AES findings showed all of them to be inhibitor resistant beta-lactamases. The antibiogram is shown in Table-1.
Susceptibility of the three isolates to amoxicillin (30[micro]g), amoxicillin/clavulanic acid (20/10[micro]g), pipercillin (100[micro]g), pipercillin/tazobactam (100/10[micro]g), cefoxitin (30[micro]g), ceftazidime (30[micro]g) was further evaluated by the Kirby Bauer disk diffusion method. (9) The phenotype amoxycillinresistant, amoxycillin plus clavulanate-resistant, pipercillin-intermediate, pipercillin/tazobactamsusceptible, cefoxitin-susceptible and ceftazidime-susceptible was observed in these isolates.
DISCUSSION: Despite the extensive use of [beta]-lactam/[beta]-lactamase inhibitor combinations in most countries, inhibitor resistant [beta]-lactamases have been more frequently isolated in Europe than in the USA, where they have been rarely reported. (10) Their occurrence have also been detected in Malaysia from E. coli isolates. (11) However, there is a paucity of information about IRT geographical distribution, probably due to inadequate phenotypic techniques using standard laboratory susceptibility tests. It was demonstrated that the automated system was able to detect 86.7% of IRT-producing E.coli. (12) Livermore DM et al. observed that for Enterobacteriaceae with IRT, the vitek 2 AES achieved 60% full and 40% partial agreement with the genotypic analysis, and penicillinases were considered as the alternative cause for the partial agreement strains. (13) However, the characterization must be completed by iso-electric points of [beta]-lactamases, determination of kinetic parameters and the use of molecular biology techniques. (14)
These variants of [beta]-lactamases occur among both community and hospital-acquired isolates. They have been recovered from urine (Predominantly), blood, sputum and stool. (8) So far, only one isolate has been reported from an agricultural source. (15)
In our cases, the Vitek 2 showed concordant result with the disk diffusion method but the characterisation could not be carried out due to lack of infrastructure of molecular techniques in our institute. These isolates could be IRT or OXA enzymes or cephalosporinase hyperproduction. Despite our inability to confirm the findings, we report these cases to show the possibility of strains possessing these enzymes in Manipur and they were susceptible to ceftriaxone, cefepime, cefuroxime, imipenem, meropenem, ertapenem, colistin, amikacin, gentamicin and pipercillin-tazobactam.
CONCLUSION: The occurrence of inhibitor resistant [beta]-lactamases has been under-reported from this region. The reasons being, unreliable detection by the routine susceptibility tests, lack of automation like Vitek2 which could at least help in preliminary detection or molecular biology methods for confirmation in most institutes. In view of inadequate data regarding the incidence of these beta-lactamases in our region, we report these cases.
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Rajkumar Manojkumar Singh , Mutum Usharani Devi , L. Kokindro Singh , Huidrom Lokhendro Singh 
[1.] Rajkumar Manojkumar Singh
[2.] Mutum Usharani Devi
[3.] L. Kokindro Singh
[4.] Huidrom Lokhendro Singh
PARTICULARS OF CONTRIBUTORS:
[1.] Assistant Professor, Department of Microbiology, Jawaharlal Nehru Institute of Medical Sciences, Imphal.
[2.] Associate Professor, Department of Microbiology, Jawaharlal Nehru Institute of Medical Sciences, Imphal.
[3.] Assistant Professor, Department of Microbiology, Jawaharlal Nehru Institute of Medical Sciences, Imphal.
FINANCIAL OR OTHER COMPETING INTERESTS: None
[4.] Professor, Department of Microbiology, Jawaharlal Nehru Institute of Medical Sciences, Imphal.
NAME ADDRESS EMAIL ID OF THE CORRESPONDING AUTHOR:
Dr. Rajkumar Manojkumar Singh, Department of Microbiology, Jawaharlal Nehru Institute of Medical Sciences, Imphal-795005, Manipur, India.
Date of Submission: 05/06/2015.
Date of Peer Review: 06/06/2015.
Date of Acceptance: 20/06/2015.
Date of Publishing: 27/06/2015.
Table 1: Susceptibility reports Antimicrobial Case 1 MIC Inter- pretation Ampicillin [greater than R or equal to] 32 Amoxicillin/ Clavulanic [greater than R acid or equal to]32 Piperacillin/Tazobactam [less than or S equal to]4 Cefuroxime [less than or S equal to]4 Ceftriaxone [less than or S equal to]1 Cefepime [less than or S equal to]1 Cefoperazone/ Sulbactam [greater than R or equal to]64 Ertapenem [less than or S equal to]0.5 Imipenem [less than or S equal to]1 Meropenem [less than or S equal to]0.5 Amikacin [less than or S equal to]2 Gentamicin [less than or S equal to]1 Ciprofloxacin [greater than R or equal to]4 Nalidixic acid [greater than R or equal to]32 Tigecycline [less than or S equal to]0.5 Nitrofurantoin [less than or S equal to]16 Trimethoprim/ [greater than R Sulfamethoxazole or equal to]320 Colistin [less than or S equal to]0.5 Antimicrobial Case 2 MIC Inter- pretation Ampicillin [greater than R or equal to]32 Amoxicillin/ Clavulanic [greater than R acid or equal to]32 Piperacillin/Tazobactam 64 I Cefuroxime [less than or S equal to]4 Ceftriaxone [less than or S equal to]1 Cefepime [less than or equa S Cefoperazone/ Sulbactam [greater than R or equal to]64 Ertapenem [less than or S equal to]0.5 Imipenem [less than or S equal to]1 Meropenem [less than or S equal to]0.25 Amikacin [less than or S equal to]2 Gentamicin [less than or S equal to]1 Ciprofloxacin 2 I Nalidixic acid 2 S Tigecycline [less than or S equal to]0.5 Nitrofurantoin 64 I Trimethoprim/ [less than or S Sulfamethoxazole equal to]20 Colistin [less than or S equal to]0.5 Antimicrobial Case 3 MIC Inter- pretation Ampicillin [greater than R or equal to]32 Amoxicillin/ Clavulanic [greater than R acid or equal to]32 Piperacillin/Tazobactam 8 S Cefuroxime 4 S Ceftriaxone 2 S Cefepime [less than or S equal to]1 Cefoperazone/ Sulbactam [greater than R or equal to]64 Ertapenem 0.5 S Imipenem 1 S Meropenem [less than or S equal to]0.5 Amikacin [less than or S equal to]2 Gentamicin [less than or S equal to]1 Ciprofloxacin 2 I Nalidixic acid [greater than R or equal to]32 Tigecycline [less than or S equal to]1 Nitrofurantoin 32 S Trimethoprim/ 80 R Sulfamethoxazole Colistin [less than or S equal to]0.5 S-Sensitive, I-Intermediate, R-Resistant
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|Title Annotation:||CASE REPORT|
|Author:||Singh, Rajkumar Manojkumar; Devi, Mutum Usharani; Singh, L. Kokindro; Singh, Huidrom Lokhendro|
|Publication:||Journal of Evolution of Medical and Dental Sciences|
|Date:||Jun 29, 2015|
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