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Observations on Carbapenem resistance by minimum inhibitory concentration in Nosocomial isolates of Acinetobacter species: an experience at a Tertiary Care Hospital in North India.

ABSTRACT

Acinetobacter species are emerging as an important nosocomial pathogen. Multidrug-resistant Acinetobacter spp. has limited the option for effective treatment. Although carbapenems are effective for the treatment of such infections, resistance to this drug has recently been reported. This study was undertaken to assess resistance to carbapenem in clinical isolates of Acinetobacter spp. from hospitalized patients by both discdiffusion and minimum inhibitory concentration (MIC) methods. All clinical samples from suspected cases of nosocomial infections were processed, and 265 isolates were identified as Acinetobacter species. These isolates were tested for antibiotic resistance by the disc-diffusion method with 14 antimicrobials, including meropenem and imipenem. Thereafter, all Acinetobacter species were subjected to MIC for meropenem. More than 80% resistance to second- and third-generation cephalosporins, aminoglycosides, and quinolones was recorded. Thirty percent of the strains were resistant to cefoperazone/sulbactam. Resistance to meropenem was observed in 6.4% of Acinetobacter spp. while 8.3% of the isolates showed intermediate resistance detected by MIC. All carbapenem-resistant/intermediate strains were also resistant to other (>10) antibiotics tested by the disc-diffusion method. The rising trend of resistance to carbapenem poses an alarming threat to the treatment for such infections. Regular monitoring, judicious prescription, and early detection of resistance to carbapenem are necessary to check further dissemination of drug resistance in Acinetobacter spp.

Key words: Acinetobacter; Acinetobacter baumannii; Antibiotic resistance; Carbapenem; Cross-infections; Drug resistance, Microbial; Meropenem; India

INTRODUCTION

Although genus Acinetobacter was originally identified in the early 20th century, it was recognized as a ubiquitous pathogen only in the last decade (1). Acinetobacter baumannii, a member of the Acinetobacter calcoaceticus--A. baumannii complex, makes up to 73% of all Acinetobacter spp. and is the most commonly-involved pathogen in clinical infections (2). During the last decade, hospital-acquired infections involving multidrug-resistant A. baumannii isolates have been reported, often in association with contamination of hospital equipment or cross-contamination by colonized hands of personnel attending patients (1). Initial concern about multidrug-resistant and carbapenem-resistant Acinetobacter baumannii (CRAB)-associated infections began when the first hospitalwide outbreak occurred in New York city in 1991 (3). Since then, reports of CRAB have been accumulating from other parts of the world (4), including India (5). Currently, the spread in hospital populations of resistant microorganisms is of great concern worldwide, suggesting that we may be approaching the post-antimicrobial era (6). This study was undertaken to assess resistance to carbapenem in clinical isolates of Acinetobacter spp. from hospitalized patients by both disc-diffusion and minimum inhibitory concentration (MIC) methods.

MATERIALS AND METHODS

Duration and place of study

A three-year study (2003-2006) was conducted to determine the susceptibility of nosocomial isolates of Acinetobacter spp. to different antimicrobials, including imipenem and meropenem. Various specimens were collected from patients admitted to different wards and intensive care unit of S.S. Hospital, Banaras Hindu University, Varanasi, India.

Identification of Acinetobacter spp.

Isolation of Acinetobacter spp. was done. Briefly, all clinical specimens were initially processed to separate the oxidase-negative, non-fermenters from other gram-negative bacilli. Thereafter, identification was done to confirm Acinetobacter spp. by standard protocol (7).

In vitro susceptibility

Susceptibility to various antimicrobial agents was determined by the disc-diffusion method and MIC by the agar dilution method following the guidelines of Clinical and Laboratory Standards Institute (CLSI). Antimicrobial susceptibility testing was performed on Mueller Hinton agar by the discdiffusion method for the following antimicrobial agents (Hi-Media, Mumbai, India) with their concentration given in parentheses: cefotaxime (30 ig), ceftazidime (30 ig), cefoperazone (75 ig), ciprofloxacin (05 ig), norfloxacin (10 ig), amikacin (30 ig), gentamicin (10 ig), tobramycin (10 ig), netilmicin (30 ig), piperacillin (100 ig), carbenicillin (100 ig), cefoperazone/sulbactam (75 ig/30 ig), meropenem (10 ig), and imipenem (10 ig) by the Kirby-Bauer method. Further in vitro susceptibility was determined for meropenem (AstraZeneca, India) by MIC with the agar dilution method, and results were interpreted according to the guidelines of CLSI ([less than or equal to] 4 [micro]g/mL=sensitive, 8 [micro]g/ mL=Intermediate, and [greater than or equal to] 16 [micro]g/mL=resistant). Quality control of susceptibility testing was done using ATCC 27853 Pseudomonas aeruginosa.

RESULTS

In total, 265 Acinetobacter spp. were isolated from 1,242 culture-positive samples from hospitalized patients and were identified up to species level as A. baumannii (91%) and A. Iwoffii (9%). On performing disc-diffusion for antimicrobial susceptibility, Acinetobacter spp. showed more than 80% resistance to third-generation cephalosporins. Among quinolones, 81% of the isolates were resistant to ciprofloxacin, while norfloxacin was inactive in 78% cases of nosocomial urinary tract infection (UTI) caused by Acinetobacter spp. Among aminoglycosides, although amikacin was relatively effective, still 74% of Acinetobacter spp. showed resistance to it. Cefoperazone/ sulbactam combination was effective with an overall resistance of 31% while 98% of Acinetobacter spp. were resistant to piperacillin. Among carbapenems, 9.1% of the isolates were resistant to imipenem and 9.8% to meropenem (Table 1).

On performing MIC, 39 isolates of Acinetobacter spp., which were resistant to meropenem, showed 6.4% and 8.3% of absolute and intermediate resistance respectively. These 39 isolates were recovered from 34 patients whose clinical data revealed that most of these isolates were from patients admitted to intensive care units (Table 2). On further analysis, it was observed that 44.9% of the isolates were on borderline to the moderate/resistance range (Fig.). Interestingly, all carbapenem-resistant/intermediate strains of Acinetobacter spp. were also resistant to 12 other antibiotics tested by the disc-diffusion method.

DISCUSSION

In the present study, an overall 18% isolation of Acinetobacter species in nosocomial colonization/ infections was observed. Acinetobacter species accounted for 1.4% of all nosocomial infections during 1971-1981 in a university hospital in the United States (8). A more recent study in a university hospital found that hospitalization in an intensive care unit and previous administration of antibiotics were associated with Acinetobacter colonization at various sites of the body in 3.2-10.8 per 1,000 patients (9). Contrary to the previous studies, a higher prevalence of Acinetobacter spp. in the region could be due to lack of good infection-control practices, personal hygiene, over-crowding situations in infirmary, and heavy patient load.

In this study, more than 75% of the isolates were resistant to third-generation cephalosporins, aminoglycosides, and quinolones. Other studies on Acinetobacters have depicted similar results with respect to these antibiotics (10-13). Thirty-one percent of these isolates was resistant to cefoperazonesulbactam; the efficacy of this drug was significant (p<0.001) compared to other groups of antimicrobials. However, another study showed 46% resistance to cefoperazone-sulbactam by the disc-diffusion method (11).

Resistance to meropenem was observed in 9.8% of Acinetobacter spp. by the disc-diffusion method while 6.4% and 8.3% of the isolates were resistant and intermediate respectively by MIC. Till date, there are limited reports from India on resistance to carbapenem, confirmed by MIC, in the nosocomial isolates of Acinetobacter species (5,14). Taneja et al. reported a high incidence (>20%) of resistance to carbapenem among Acinetobacters in India. However, a report from France showed that 17% of Acinetobacter spp. was resistant to meropenem by the agar dilution method while a study in the UK reported 10% resistance which is quite similar to our results (15,16).

Other studies have shown a high incidence of resistance to carbapenem among Acinetobacters from patients in intensive care units, suggesting that intensive care units are the epicentre for carbapenem-resistant Acinetobacters (17,18).

Meropenem-resistant Acinetobacter spp. was also found to be resistant to all other antimicrobials (Pandrug-resistant Acinetobacter baumannii) (19). This disturbing situation could be attributed to the increased use of antibiotics which has to be controlled by a strict policy for use of antibiotics, in the face of aggressive marketing by the pharmaceuticals. Effective strategies, such as strict infection-control measures, judicious prescriptions of antibiotics, antimicrobial resistance surveillance programmes, and antibiotic cycling have all been tried successfully to control drug resistance in some countries (20).

Carbapenems have become the drugs of choice in Acinetobacter-associated infections in many centres but are slowly being compromised by the emergence of carbapenem-hydrolyzing-lactamases of molecular class B and D (19). Class B carbapenemases found so far in Acinetobacters include various IMP and VIM types; class D enzymes include members of the OXA-23- and OXA-24-related families and various unsequenced types (20). Loss of porins, PBP with reduced affinity, efflux pump, AmpC, and different class B and D [beta]-lactamases have been associated with resistance to carbapenems in clinical strains of Acinetobacter spp. (21,22). A report from India on mechanisms of carbapenem resistance (phenotypic method) among Acinetobacters has suggested that AmpC is responsible for such resistance (5).

Despite the low prevalence of carbapenem resistance in this study, caution has to be exercised in its use in critically-ill hospitalized patients to check any further increase in the resistance to carbapenems. It is notable that almost 45% isolates of Acinetobacter species were on the borderline to moderate/resistant range to carbapenem. Regular monitoring and documentation of carbapenem resistance is, therefore, crucial while developing strategies to control infections due to Acinetobacter spp. in hospitalized patients.

ACKNOWLEDGEMENTS

The authors are grateful to the Indian Council of Medical Research, New Delhi, for extending financial support to conduct the present study. The authors do not directly own any stock/share in a company that might be financially affected by the conclusion of this article.

REFERENCES

(1.) Bergogne-Berezin E, Towner KJ. Acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological features. Clin Microbiol Rev 1996;9:148-65.

(2.) Bouvet PJM, Grimont PAD. Taxonomy of the genus Acinetobacter with the recognition of Acinetobacter baumannii sp. nov., Acinetobacter haemolyticus sp. nov., Acinetobacter johnsonii sp. nov., Acinetobacter junii sp. nov., and emended descriptions of Acinetobacter calcoaceticus and Acinetobacter lwoffii. Int J Syst Bacteriol 1986;36:228-40.

(3.) Urban C, Go E, Mariano N, Berger BJ, Avraham I, Rubin D, Rahal JJ. Effect of sulbactam on infections caused by imipenem-resistant Acinetobacter calcoaceticus biotype anitratus. J Infect Dis 1993;167:448-51.

(4.) Bou G, Cervero G, Dominguez MA, Quereda C, Martinez-Beltran J. Characterization of a nosocomial outbreak caused by a multiresistant Acinetobacter baumannii strain with a carbapenem-hydrolyzing enzyme: high-level carbapenem resistance in A. baumannii is not due solely to the presence of beta-lactamases. J Clin Microbiol 2000;38:3299-305.

(5.) Sinha M, Srinivasa H. Mechanisms of resistance to carbapenems in meropenem resistant Acinetobacter isolates from clinical samples. Indian J Med Microbiol 2007;25:121-5.

(6.) Acar JF, Goldstein FW. Consequences of increasing resistance to antimicrobial agents. Clin Infect Dis 1998;27(Suppl 1):S125-S30.

(7.) Baron EJ, Peterson LR, Finegold SM. Genus Acinetobacter. In: Non-fermentative Gram-negative bacilli and coccobacilli. Bailey and Scott's diagnostic microbiology. 9th ed. St. Louis, MO: Mosby, 1994:401-2.

(8.) Larson E. A decade of nosocomial Acinetobacter. Am J Infect Control 1984;12:14-8.

(9.) Struelens MJ, Carlier E, Maes N, Serruys E, Quint WG, van Belkum A. Nosocomial colonization and infection with multiresistant Acinetobacter baumannii: outbreak delineation using DNA macrorestriction analysis and PCR-fingerprinting. J Hosp Infect 1993; 25:15-32.

(10.) Prashanth K, Badrinath S. In vitro susceptibility pattern of acinetobacter species to commonly used cepha losporins, quinolones and aminoglycosides. Indian J Med Microbiol 2004;22:97-103.

(11.) Kucukates E, Kocazeybek B. High resistance rate against 15 different antibiotics in aerobic gram-negative bacteria isolates of cardiology intensive care unit patients. Indian J Med Microbiol 2002;20:208-10.

(12.) Anstey NM, Currie BJ, Withnall KM. Community-acquired Acinetobacter pneumonia in the Northern Territory of Australia. Clin Infect Dis 1992;14:83-91.

(13.) Singh AK, Sen MR, Anupurba S, Bhattacharya P. Antibiotic sensitivity pattern of the bacteria isolated from nosocomial infections in ICU. J Commun Dis 2002;34:257-63.

(14.) Taneja N, Maharwal S, Sharma M. Imipenem resistance in nonfermenters causing nosocomial urinary tract infections. Indian J Med Sci 2003;57:294-9.

(15.) Aubert G, Guichard D, Vedel G. In-vitro activity of cephalosporins alone and combined with sulbactam against various strains of Acinetobacter baumannii with different antibiotic resistance profiles. J Antimicrob Chemother 1996;37:155-60.

(16.) Henwood CJ, Gatward T, Warner M, James D, Stockdale MW, Spence RP et al. Antibiotic resistance among clinical isolates of Acinetobacter in the UK, and in vitro evaluation of tigecycline (GAR-936). J Antimicrob Chemother 2002;49:479-87.

(17.) Manikal VM, Landman D, Saurina G, Oydna E, Lal H, Quale J. Endemic carbapenem-resistant Acinetobacter species in Brooklyn, New York: citywide prevalence, interinstitutional spread, and relation to antibiotic usage. Clin Infect Dis 2000;31:101-6.

(18.) Corbella X, Montero A, Pujol, M, Dominguez MA, Ayats J, Argerich MJ et al. Emergence and rapid spread of carbapenem resistance during large and sustained hospital outbreak of multiresistant Acinetobacter baumannii. J Clin Microbiol 2000;38:4086-95.

(19.) Go ES, Urban C, Burns J, Kreiswirth B, Eisner W, Mariano N et al. Clinical and molecular epidemiology of acinetobacter infections sensitive only to polymyxin B and sulbactam. Lancet 1994;344:1329-32.

(20.) Merz LR, Warren DK, Kollef MH, Fraser VJ. Effects of an antibiotic cycling program on antibiotic prescribing practices in an intensive care unit. Antimicrob Agents Chemother 2004;48:2861-5.

(21.) Livermore DM. The impact of carbapenemases on antimicrobial development and therapy. Curr Opin Investing Drugs 2002;3:218-24.

(22.) Poirel L, Nordmann P. Acquired carbapenem-hydrolyzing beta-lactamases and their genetic support. Curr Pharm Biotechnol 2002;3:117-27.

Correspondence and reprint requests should be addressed to:

Dr. T.M. Mohapatra

Professor, Department of Microbiology

Institute of Medical Science

Banaras Hindu University

Varanasi (UP)

India

Email: tmmohapatra2000@yahoo.com

A. Gaur, A Garg, P. Prakash, S. Anupurba, and T.M. Mohapatra

Department of Microbiology, Institute of Medical Science, Banaras Hindu University, Varanasi (UP) 221 005, India
Table 1. Antibiotic resistance pattern of Acinetobacter species
isolated from different wards, expressed in percentage (%)

                     Post-opera-
                   tive and others    ICU                    Overall
Antibiotic             (n=154)       (n=89)   Burns (n=22)   (n=265)

[beta]-lactams
  Piperacillin          97.9          97.4       100.0        97.9
  Carbenicillin         69.6          71.4        50.0        68.8
  Cefotaxime            79.0          83.5        83.3        80.8
  Ceftazidime           77.6          83.5        83.3        80.0
  Cefoperazone          79.0          87.3        77.8        82.3
  Imipenem              07.1          12.3        09.1        09.1
  Meropenem             07.7          12.7        11.1        09.8
Aminoglycosides
  Gentamicin            83.9          87.3        94.4        85.8
  Tobramycin            82.5          86.0        88.9        84.2
  Amikacin              74.8          73.4        77.8        74.6
  Netilmicin            78.3          83.5        83.3        80.4
Quinolones
  Ciprofloxacin         79.7          83.5        77.8        80.8
  Norfloxacin           78.3          71.4       100.0        78.1
Others
  Cefoperazone +
    sulbactam           27.8          45.6        27.8        31.2

ICU=Intensive care unit

Table 2. Clinical data of patients producing carbapenem-resistant
Acinetobacters

Sl.                         Age
no.   Sample     Ward/ICU   (years)    Sex   Clinical diagnosis

1     Pus        FSW        45         F     Gall bladder perforation
2     Pus        MSW        34         M     Non-healing ulcer
3     Pus        MSW        33         F     Polytrauma
4     Pus        Spl        50         M     Diabetic foot
5     Blood      NICU        1         M     Neonatal septicaemia
6     Pus        Tr         32         M     Crush injury
7     Pus        Ortho       7         F     Abscess Rt knee
8     Pus        MSW        33         M     Non-healing ulcer
9     Pus        Gyn        28         F     PO infection
10    Swab       ICU        26         M     Multiple fracture
11    ETT        ICU        50         F     Renal failure
12    ETT        ICU        61         M     ARDS, with PUO
13    ETT        ICU        21         F     Respiratory failure
14    Urine      NICU       12 days    M     UTI
15    ETT        ICU        35         M     COPD
16    Pus        MSW        55         M     Necrotizing fascitis
17    Pus        ICU        45         M     Road traffic accident
18    Pus        FSW        35         M     Abdominal surgery
19    Pus        MSW        41         M     Laparotomy
20    Pus        Burns      70         M     90% burn
21    Pus        NICU        1 month   M     Cellulitis
22    ETT        ICU        10         F     Head injury
23    ETT        ICU        35         M     Bronchial asthma
24    Tr. tube   ICU        30         M     Pneumonia
25    Pus        CTVS       45         M     Infective endocarditis
26    Pus        Burns      30         F     70% burn
27    Urine      Gyn        22         F     PO infection
28    ETT        ICU        32         M     COPD, complications
29    Pus        MSW        44         M     Abdominal surgery
30    Tr. tube   ICU        43         F     Bronchial asthma
31    ETT        ICU        70         M     Pneumonia
32    Pus        Spl        48         M     Bracheal artey injury
33    Urine      ICU        29         M     Laparatomy
34    Pus        Surg       27         F     Deglobing injury scalp
35    Cat. tip   ICU        48         F     Opium poisoning
36    ETT        ICU        35         F     GI bleeding, pneumonia
37    Swab       ICU        22         M     Renal failure
38    ETT        ICU        43         F     Pneumonia
39    Blood      ICU        47         M     Septicaemia

ARDS=Acute respiratory distress syndrome; Cat=Catheter; COPD=Chronic
obstructive pulmonary disease; CTVS=Cardiovascular thoracic surgery;
ETT=Endotracheal tube; FSW=Female surgical ward; GI=Gastrointestinal;
Gyn=Gynaecology; ICU=Intensive care unit; MSW=Male surgical ward,
NICU=Neonatal ICU, PO=Postoperative; Ortho=Orthopaedics; PUO=Pyrexia
of unknown origin; Sp1=Special ward; Tr=Tracheostomy; UTI=Urinary
tract infection

Fig. Response of Acinobacter spp. to various
concentration ranges of meropenem by MIC

Meropenem concentration range ([micro]g/ml)

    Range   No. of isolates

1   0.25         49
2   0.5          29
3   1            76
4   2            43
5   4            29
6   8            22
7   16           11
8   32            6

MIC=Minimum inhibitory concentration

Note: Table made from bar graph.
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Article Details
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Author:Gaur, A.; Garg, A.; Prakash, P.; Anupurba, S.; Mohapatra, T.M.
Publication:Journal of Health Population and Nutrition
Article Type:Clinical report
Geographic Code:9INDI
Date:Jun 1, 2008
Words:2816
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