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Detection of inhibitor resistant beta-lactamases in a tertiary care centre of Manipur, India: a report of three cases.

INTRODUCTION: The synergistic effect of [beta]-lactamase inactivators (Clavulanic acid, sulbactam and tazobactam) and a penicillin or cephalosporin has been employed to combat [beta]-lactamases. (1) Reports have established regarding the development of resistance to these inhibitors which might be caused by the hyperproduction of unmodified TEM(TEM named after the patient, Temoniera, from whom it was isolated)-type [beta]-lactamase, or modification of the outer membrane proteins, or production of OXA (Oxacillinase)-type enzymes, or hyperproduction of cephalosporinases. (2-6) Since 1990, the emergence of inhibitor-resistant TEM [beta]-lactamases (IRT) has further compromised the effect of [beta]- lactamase inhibitors. (7)

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.

DOI: 10.14260/jemds/2015/1323

REFERENCES:

(1.) Chaibi EB, Sirot D, Paul G, Labia R. Inhibitor-resistant TEM [beta]-lactamases: phenotypic, genetic and biochemical characteristics. J Antimicrob Chemother 1999; 43:447-458.

(2.) Martinez JL, Vicente MF, Delgado-Iribarren A, Perez-Diaz JC, Baquero F. Small plasmids are involved in amoxycillin-clavulanate resistance in Escherichia coli. Antimicrob Agents Chemother 1989; 33:595.

(3.) Wu PJ, Shannon K, Phillips I. Effect of hyper production of TEM-1[beta]-lactamase on in vitro susceptibility of Escherichia coli to [beta]-lactam antibiotics. Antimicrob Agents Chemother 1994; 38: 494-498.

(4.) Wu PJ, Shannon K, Phillips I. Mechanisms of hyper production of TEM-1 [beta]-lactamase by clinical isolates of Escherichia coli. J Antimicrob Chemother 1995; 36: 927-939.

(5.) Reguera JA, Baquero F, Perez-Diaz JC, Martinez JL. Factors determining resistance to [beta]-lactam combined with [beta]-lactamase inhibitors in Escherichia coli. J Antimicrob Chemother 1991; 27: 569-575.

(6.) Bergstrom S, Normark S. [beta]-lactam resistance in clinical isolates of Escherichia coli caused by elevated production of the AmpC-mediated chromosomal [beta]-lactamase. Antimicrob Agents Chemother 1979; 16: 427-433.

(7.) Vedel G, Belaaouaj A, Gilly L, Labia R, Philippon A, Nevot P. Clinical isolates of Escherichia coli producing TRI [beta]-lactamases: novel TEM-enzymes conferring resistance to [beta]-lactamase inhibitors. J Antimicrob Chemother 1992; 30: 449-462.

(8.) Bonomo RA, Rice LB. Inhibitor resistant class A beta-lactamaes. Front Biosci 1999; 4: 34-41.

(9.) Canto'n R, Morosini MI, Martin O, de la Maza S, de la Pedrosa EG. IRT and CMT [beta]-lactamases and inhibitor resistance. Clin Microbiol Infect 2008; 14(Suppl. 1): 53-62

(10.) Kaye KS, Gold HS, Schwaber MJ et al. Variety of b-lactamases produced by amoxicillin-clavulanate-resistant Escherichia coli isolated in the Northeastern United States. Antimicrob Agents Chemother 2004; 48: 1520-1525.

(11.) Wong JS, Mohd Azri ZA, Subramaniam G, Ho SE, Palasubramaniam S, Navaratnam P. [beta]-lactam resistance phenotype determination in Escherichia coli isolates from University Malaya Medical Centre. Malays J Pathol 2003; 25: 113-119.

(12.) Bret L, Peyret M, Romaszko JP, Henquell C, Sirot D, Chanal C et al. Detection of inhibitor-resistant TEM [beta]-lactamases(IRT) on ATB Plus system. In Abstracts of the Seventh European Congress of Clinical Microbiology and Infectious Diseases, Vienna. Abstract 167. Excepta Medica Medical Communications, Vienna, Austria, 1995.

(13.) Livermore DM, Struelens M, Amorim J, Baquero F, Bille J, Canton R, Henning S, Gatermann S, Marchese A. Multicentric evaluation of the Vitek2 Advanced expert System for interpretive reading of antimicrobial resistance tests. J Antimicrob Chemother. 2002; 49: 289-300.

(14.) Thomson CJ, Amyes SGB. Back mutations to the TEM-1 [beta]-lactamase from TRC-1 lead to restored sensitivity to clavulanic acid. J Med Microbiol 1995; 42: 429-432.

(15.) Hunter J, Corkill JE, Lennan AG, Fletcher JN, Hart CA. Plasmid encoded beta-lactamases resistant to inhibition by clavulanic acid produced by calf faecal coliforms.. Research in Veterinary Science 1995; 55: 367-370.

Rajkumar Manojkumar Singh [1], Mutum Usharani Devi [2], L. Kokindro Singh [3], Huidrom Lokhendro Singh [4]

AUTHORS:

[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.

E-mail: rkmksingh@gmail.com

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
Geographic Code:9INDI
Date:Jun 29, 2015
Words:2105
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