| Abstract|| |
Context: Bacterial isolates from intra-abdominal infections, in particular, peritonitis and their unpredictable antimicrobial resistance patterns, continue to be a matter of concern not only globally but regionally too. Aim: An attempt in the present study was made to study the patterns of drug resistance in bacterial isolates, especially gram negative bacilli in intra-abdominal infections (IAI) in our hospital. Materials and Methods: From 100 cases of peritonitis, identification of isolates was done as per recommended methods. Antimicrobial susceptibility and extended-spectrum beta-lactamase (ESBL) testing were performed following the CLSI guidelines. Results: A total of 133 clinical isolates were obtained, of which 108 were aerobes and 22 anaerobes. Fungal isolates were recovered in only three cases. Escherichia coli (47/108) emerged as the most predominant pathogen followed by Klebsiella spp. (27/108), while Bacteroides fragilis emerged as the predominant anaerobe (12/22). Among coliforms, 61.7% E. coli and 74.1% Klebsiella spp. were ESBL positive. A high level of resistance was observed for beta lactams, ciprofloxacin, amikacin, and ertapenem. Ertapenem resistance (30-41%) seen in coliforms, appears as an important issue. Imipenem, tigecycline, and colistin were the most consistently active agents tested against ESBL producers. Conclusion: Drug resistance continues to be a major concern in isolates from intra-abdominal infections. Treatment with appropriate antibiotics preceded by antimicrobial resistance testing aided by early diagnosis, adequate surgical management, and knowledge of antibiotic - resistant organisms appears effective in reducing morbidity and mortality in IAI cases.
Keywords: Antibiotic resistance, ESBL, gram negative bacilli, intra-abdominal infections.
|How to cite this article:|
Shree N, Arora BS, Mohil RS, Kasana D, Biswal I. Bacterial profile and patterns of antimicrobial drug resistance in intra-abdominal infections: Current experience in a teaching hospital. Indian J Pathol Microbiol 2013;56:388-92
|How to cite this URL:|
Shree N, Arora BS, Mohil RS, Kasana D, Biswal I. Bacterial profile and patterns of antimicrobial drug resistance in intra-abdominal infections: Current experience in a teaching hospital. Indian J Pathol Microbiol [serial online] 2013 [cited 2020 Feb 20];56:388-92. Available from: http://www.ijpmonline.org/text.asp?2013/56/4/388/125321
| Introduction|| |
Generalized peritonitis due to intra-abdominal infections (IAIs), despite advances in surgical techniques, comprise more than 25% of emergency operations in our hospitals.  Decline in morbidity and mortality in IAI is a welcome observation in Indian scenario.  Knowing the prevalent pattern of antimicrobial resistance is an important issue especially when gram negative isolates continue to exhibit rampant resistance to various currently in use antimicrobial agents.  Therefore, empirical selection of the right kind of anti-microbial therapy is significant in reducing morbidity and mortality. In Indian context, data especially antimicrobial resistance status is still scarce for this very common problem.
| Materials and Methods|| |
A total of 100 cases of intra-abdominal sepsis were studied in department of Microbiology and General Surgery of our hospital from November, 2010 to January, 2012 (15 months). A total of 125 clinical specimens (Pus/exudates, 0.5 ml minimum and blood, 10 ml) from 100 cases of intra-abdominal sepsis were collected aseptically in Robertson Cooked Meat (RCM) medium from patients presenting with clinical picture of acute intra-abdominal sepsis and subjected to culture and isolation for clinically significant isolates as per the criteria laid down in Mackie and McCartney, 14th edition  and Wadsworth manual of anaerobic bacteriology.  The majority of specimens were obtained during surgery, though some specimens were also taken post-operatively from cases of burst abdomen. The specimens were processed for isolation of aerobic and anaerobic bacteria by using standard microbiological techniques and their antimicrobial susceptibility was determined by using Kirby-Bauer disk diffusion technique recommended by Clinical & Laboratory Standards Institute (CLSI), 2010 guidelines.  For antimicrobial susceptibility testing quality control was performed using Escherichia More Details coli ATCC 25922; Klebsiella pneumoniae 700603 extended - spectrum beta-lactamase (ESBL)- positive control strain), and Pseudomonas aeruginosa ATCC 25843 strains.
Testing of ESBL
ESBL detection was done by phenotypic method as per CLSI (2010) recommended clavulanate combined disk and double disk synergy test using cefotaxime (30 μg), ceftazidime (30 μg) and combination of cefotaxime (30 μg)-/ceftazidime (30 μg) and clavulanic acid (10 μg). The zone diameter given by the disks with clavulanate was ≥5 mm or larger than those without the inhibitor if an ESBL was produced.
The results were tabulated and analyzed by SPSS 16 software using the chi-square (χ2 )-tests. A 'P' value of less than 0.05 was taken as significant. The study was approved by Ethical Committee of our hospital.
| Results|| |
A total of 108 strains of aerobic bacteria and 22 strains of anaerobic bacteria (either as sole pathogen or in association with other organisms) were isolated during the present study. Three Candida spp. were also isolated. The 92 aerobic Gram negative bacilli (GNB) isolates comprised of 47 (43.5%) E. coli, Klebsiella spp. 27 (25.4%), Acinetobacter spp. 08 (7.4%), P. aeruginosa 06 (5.5%), Enterobacter spp 02 (1.8%), Citrobacter spp. 01 (0.9%) and Proteus vulgaris 01 (0.9%). The most commonly isolated organism was E. coli, of which 61.7% of the isolates (n = 29) were ESBL positive. Among Klebsiella isolates, 74.9 % (n = 20) were ESBL positive. [Table 1] illustrates the distribution of aerobes and anaerobes (including Candida spp.) and their antimicrobial resistance pattern.
|Table 1: Resistance rates (%) of various gram negati ve non-duplicate clinical isolates|
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The antimicrobial resistance levels of E. coli, and Klebsiella spp. to the carbapenems ranged from 29% to 41% (ertapenem), 14% to 15% (meropenem), and nil toward imipenem. Imipenem, tigecycline, and colistin remained the most consistently active against all gram negative pathogens. Susceptibility to other commonly used antibiotics including cephalosporins, fluoroquinolones, aminoglycosides, and carbapenems also showed a decline. Resistance levels were higher for the ESBL-producing organisms [Table 2]. A high level of resistance among gram negative organisms to third generation cephalosporin antibiotics as well as β lactam/β lactamase inhibitors is also evident [Figure 1]. Resistance rates for E. coli strains were as high as 78.7% for ciprofloxacin, 72.3% for cefotaxime, 25.5% for amikacin, 46.8% for netilmicin, 28% for piperacillin/tazobactam combination, 32% for cefoperazone/sulbactam combination, 30% and 15% for ertapenem and meropenem, respectively.
|Table 2: Frequency of ESBL* producti on in Escherichia coli siella spp. in IAI|
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Sensitivity patterns of Klebsiella spp. also revealed low susceptibility for various antimicrobials with a resistance rate of 81.5% for cefotaxime, 66.7% for ciprofloxacin, 41%, 48%, 47%, 37% & 41% & 15% respectively for amikacin, netilmicin, piperacillin/tazobactam combination, cefoperazone/sulbactam combination and ertapenem & meropenem. Overall, imipenem, tigecycline and colistin appeared to be the most effective (100 %) against the majority of gram negative isolates.
In P. aeruginosa, a resistance rate of 66.67% (n = 04) and 50% (n = 03) was observed for ciprofloxacin and netilmicin respectively, whereas 33.33% (n = 02) resistance was observed for amikacin and meropenem. Resistance toward aztreonam, imipenem was not seen, while colistin was, in vitro, highly effective. Eight strains of Acinetobacter spp. isolates revealed resistance rate of 62.5%, 50%, 37.5%, 25%, and 12.5% for netilmicin, amikacin, meropenem, ciprofloxacin, and piperacillin/tazobactam in a decreasing order of resistance, respectively. Nil resistance was seen for imipenem and colistin. For gram positive cocci, a high level of resistance toward penicillin, ciprofloxacin, gentamicin in staphylococcal and enterococcal spp. was observed; however, these isolates were fully susceptible to vancomycin.
| Discussion|| |
In accord with the several global and regional reports, our study also revealed E. coli as the most frequent pathogen associated with IAI. , As also reported by others, in addition to E. coli, Klebsiella spp, P. aeruginosa, and Acinetobacter spp. were among other gram negative isolates. ,, In a study by Stephen P. Hawser in 2008, from several hospitals in India (SMART study), the most frequently isolated pathogens were E. coli (62.7 %), K. pneumoniae (16.7 %), and P. aeruginosa (5.3 %) while in another similar study by Maria Virginia Villegas et al. from 10 Latin American countries, E. coli and K. pneumoniae were the most commonly isolated organisms from such IAI. 
The prevalence of multi-drug-resistant gram negative bacilli, especially ESBL producers, has increased worldwide with marked regional variations of their distribution. ,, In our study, ESBL producers were seen to the extent of 61.70% in E. coli and 74.07% in Klebsiella spp. Stephen P. Hawser et al. (2008) reported 67 % and 55 % ESBL production respectively in E. coli and K. pneumoniae isolates in a SMART study from India. Apart from SMART global surveillance programme, several other local reports have also described variable ESBL rates in India, for example, Aggarwal et al. (2008)  reported ESBL rates as 22 % in E. coli and Klebsiella pneumonia in Pune, while Sinha et al. (2008)  showed the rates as high as 64.8 % in Jaipur. Frequency of ESBL producers as low as 26% and 35% respectively in E. coli and Klebsiella spp. has been observed in Latin American countries,  while the same has been reported as 36.8% and 26.3% in E. coli and Klebsiella spp. in Asia-Pacific region.  Variations in ESBL rates as documented by these studies reinforce the prevalence of regional variations and emphasize the need for frequent studies.
For antimicrobial resistance, interestingly, in E. coli, resistance was as high as 78.7% for ciprofloxacin, 72.3% for cefotaxime but it was relatively low for piperacillin/tazobactam (28%) and cefoperazone/sulbactam (32%) combinations. Among aminoglycosides, resistance levels of 25.5% for amikacin, 46.8% for netilmicin, while 30% and 15% for carbapenems i.e. ertapenem and meropenem respectively were evident. The resistance level for ciprofloxacin and aminoglycosides were comparatively higher than that reported by other investigators such as by Sepehri G, Goldstein and Syndman and Paterson et al. These investigators reported such resistance in the range 3-23%. ,,
Klebsiella spp. was relatively more resistant to commonly used antimicrobial agents with resistance levels as high as 81.5% for cefotaxime and 66.7% for ciprofloxacin. For other antimicrobials, resistance levels as 41% for amikacin, 48% for netilmicin, 47% for piperacillin/tazobactam combination, 37% for cefoperazone/sulbactam, & 41% & 15% respectively for ertapenem & meropenem were observed in that decreasing order. Our results are in accord with several other studies that revealed an increase in resistance in Klebsiella spp. from different regions of the world, namely Chen et al., Hauser et al., Sepehri and Villages et al.,,
Imipenem, tigecycline, and colistin appeared as the most active drugs against E. coli and Klebsiella spp. (100% susceptible) which again tallies with the results of other investigators, namely Villages et al., Hauser et al., Chaudhuri et al.,,,,,, Genotypic resistance marker analyses are limitations of our study but clearly increase in resistance, whether due mainly to ESBL production or hitherto other yet unclear mechanisms with tagged clinical impact remain a potential threat in treating such infections.  Plasmids responsible for ESBL production carrying genes encoding resistance to several other structural drug classes' especially aminoglycosides and fluoroquinolones have been identified making options of treating such infections difficult and extremely limited. In addition, fast emerging reports of carbapenem-resistant isolates are making situation still worse. 
P. aeruginosa and Acinetobacter spp. are other major nosocomial pathogens which display remarkable properties of resistance to many antibiotics; therefore, their isolation is an important cause of concern. ,, Six strains of P. aeruginosa showed high resistance level of 66.67% (n = 04) and 50% (n = 03) for ciprofloxacin and netilmicin respectively, whereas 33.33% (n = 02) resistance was observed for amikacin and meropenem separately. Resistance toward aztreonam, imipenem was not seen, while colistin was highly effective. Eight strains of Acinetobacter spp. isolated revealed resistance levels of 62.5%, 50%, 37.5%, 25%, and 12.5% for netilmicin, amikacin, meropenem, ciprofloxacin, and piperacillin/tazobactam respectively in that decreasing order but resistance for imipenem and colistin was not evident. It is comparatively lesser when compared with other studies. ,, Interestingly again, a satisfactory coverage with piperacillin/tazobactam combination and imipenem against these isolates has been reported by several other workers. ,, The present study did not include screening of Amp C beta lactamases and metallo beta lactamases (MBLs) production which is an important limitation of our study. Among gram positive isolates, the number of isolates is too less to make any definitive conclusions. Only 12 strains of Staphylococcus aureus isolated in the present study revealed resistance level of 50% (n = 06) toward erythromycin, clindamycin, and ciprofloxacin (50%). In Staphylococci, emergence of methicillin-resistant Staphylococcus aureus (MRSA) in hospital settings and therapeutic challenges it poses are well recognized. The presence of MRSA narrows down the therapeutic options to treat these infections. Of the 12 Staphylococcal isolates five strains (42%) were MRSA. Among these - 3 (60%) were sensitive to vancomycin; 1 (20%) to ciprofloxacin and vancomycin separately, and 1 (20%) to gentamicin and vancomycin separately revealing the limited options for the alternative antimicrobial agents. All five strains of S. aureus were sensitive to vancomycin (100%). Rising levels of clindamycin resistance 6/12 (50%) in Staphylococcus aureus as seen in the present study appears an unhealthy sign when compared to low levels of resistance as reported in previous studies. ,, In Enterococci, an increased resistance to various antimicrobial agents was observed as also reported in other studies. , Of the only 4 strains of Enterococci isolated, 3/4 (75%) were resistant to penicillin, 2/4 (50%) to ciprofloxacin and chloramphenicol, and 1/4 (25%) to gentamicin. However, all these four strains were sensitive to vancomycin (100%). The resistance level seen in our study for S. aureus and Enterococci is comparatively higher than that reported by Montravers et al. B. fragilis is among the most common anaerobic isolate from IAIs. , Of the 12 strains isolated, antimicrobial susceptibility of 10 strains revealed that all strains were sensitive to metronidazole, ertapenem, and meropenem, and to combination of piperacillin and tazobactam. All strains, except one, were sensitive to clindamycin and augmentin. Metronidazole, ertapenem, meropenem, and piperacillin/tazobactam combination were found 100% effective. All these antibiotics can also be used, if required, as an alternative to metronidazole for treating anaerobic infections. The universal bactericidal activity of metronidazole against B. fragilis and other anaerobes is well documented albeit rare reports of metronidazole resistance. Excellent activity against Bacteroides fragilis of several antimicrobials like metronidazole, ampicillin/sulbactam, piperacillin/tazobactam, ertapenem, meropenen imipenem along with good to moderate activity of cefoxitin is indeed well documented. ,
| Conclusion|| |
The study emphasizes need for antimicrobial susceptibility testing of clinically significant isolates not only as a routine procedure but also on periodic basis, especially when no definitive resistance or susceptibility patterns are available in a given geographic location.
| Acknowledgement|| |
We are thankful to Dr.Monorama Deb, M.D., Professor and Head (Microbiology Department), and technical staff, especially Miss. Lata Kalra - Department of Microbiology, VMMC & Safdarjang hospital, New Delhi for their technical cooperation.
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Balvinder S Arora
Departments of Microbiology, Vardhman Mahavir Medical College & Safdarjang Hospital, Room No - 507, 5th Floor, College Building, New Delhi - 110 029
Source of Support: None, Conflict of Interest: None
[Table 1], [Table 2]