|Year : 2014 | Volume
| Issue : 3 | Page : 332-337
Development of antibiotic resistance in Gram negative bacilli: An eye opener
Ravinder Pal Singh1, Sonali Jain1, Parduman Singh2, Nikunj Gupta3
1 Department of Microbiology, Goldfield Institute of Medical Sciences and Research, Ballabhgarh, Faridabad, Haryana, India
2 Department of Biochemistery, Goldfield Institute of Medical Sciences and Research, Ballabhgarh, Faridabad, Haryana, India
3 Department of Microbiology, Mohan Dai Oswal Cancer Hospital, Ludhiana, Punjab, India
|Date of Web Publication||18-Mar-2014|
40 Anamika Appts, 99 I P extn, Patparganj, Delhi - 110 092
Source of Support: None, Conflict of Interest: None
Context: Antibiotic resistance is a global problem. Organisms are showing resistance to not only the conventional antibiotics but also to the higher generation drugs. This enormous amount of resistance faced is a serious threat to mankind and this is further accentuated by the fact that the antibiotic pipeline is fast drying up. We are now left with only a handful of antibiotics to deal with all infections - serious or otherwise. The present paper highlights the current scenario of drug resistance especially in nosocomial settings. Aims and Objectives: To determine the distribution of bacterial pathogens causing nosocomial infections and their antibiogram, a surveillance data from January to December 2011 was collected in Mohan Dai Oswal Hospital. A total of 1800 samples were taken of which maximum samples were urine (766) followed by blood (428) and pus (216), and so on. Settings and Design: This observational study was conducted in the microbiology department of a multispeciality hospital during January-December 2011. Materials and Methods: A total of 1800 samples from different sources were included in the study like pus, blood, urine, sputum, etc., which were taken from patients admitted in the hospital for more than a week. Gram negative bacilli were isolated, identified, and subjected to antibiotic sensitivity test. Statistical Analysis Used: Descriptive statistics using percentages. Results: Out of the total 1800 samples included, maximum positivity was found in the pus samples (70%). Extended-spectrum beta-lactamase (ESBL) positivity was also maximum in the pus samples (90%). These ESBL positive organisms were further subjected to antibiotic sensitivity tests and huge amounts of resistance was noted to the conventional drugs including the higher end agents like Carbapenems. In light of this, newer drugs like Tigecycline, Colistin, and Polymyxin B were also tested. Barring Tigecycline, none showed favorable results. A noteworthy finding was the sensitivity of the urinary ESBL isolates to Nitrofurantoin. Conclusions: The situation is quite dangerous. The time is not far when we will be back in the dark ages of the preantibiotic era. The need of the hour is to be alert of the gravity of this situation and take necessary measures to halt its progress.
Keywords: Antibiotic resistance, Extended-spectrum betalactamase, nosocomial infections
|How to cite this article:|
Singh RP, Jain S, Singh P, Gupta N. Development of antibiotic resistance in Gram negative bacilli: An eye opener. Med J DY Patil Univ 2014;7:332-7
|How to cite this URL:|
Singh RP, Jain S, Singh P, Gupta N. Development of antibiotic resistance in Gram negative bacilli: An eye opener. Med J DY Patil Univ [serial online] 2014 [cited 2021 Dec 3];7:332-7. Available from: https://www.mjdrdypu.org/text.asp?2014/7/3/332/128976
| Introduction|| |
Microbes are remarkably adaptable and amazingly versatile. Through the course of evolution, they have developed sophisticated mechanisms for preserving genetic information and disseminating it efficiently in the interests of their survival. They recognize no boundaries. Antibiotic resistance is a global problem. The resistance developing in one part of the country, or indeed in the world, can be disseminated readily. 
For the past few decades, incidence of resistance among both the Gram negative and Gram positive organisms has reached a very high level, leading to more and more cases of treatment failure. The alarming increase in resistance is leading us to a point of no return - a day when no antimicrobial agent would be available to treat infections.
Nosocomial infections are more notorious than community-acquired infections. These infections are favored by the hospital environment. They are generally transmitted when hospital officials become complacent and the staff does not comply with the standard hygiene practices. Among the categories of bacteria that are common to infect admitted patients are extended spectrum beta-lactamases (ESBLs), vancomycin-resistant Enterococci (VRE), and methicillin-resistant Staphylococcus aureus (MRSA) since they are omnipresent in the hospital environment.
The genes coding for these are present on plasmids, which also carry genes for multidrug resistance. Similarly VRE and MRSA are also multidrug resistant (MDR). The factors favoring colonization of these organisms are numerous including low immune status, IV catheters, diabetes,  age more than 65 years, long hospital and intensive care unit (ICU) stay, and so on. , Previously, patients infected with ESBLs were found mainly in ICUs but now they are found in other wards as well. ,, Further, the combination of Amp C enzyme along with ESBLs often compound the resistance so much that the organisms are now resistant to even beta lactam-beta lactamase inhibitor combination, cephamycins and the life saving carbapenams. 
So far as Gram positive organisms are concerned, few options are still available and some antibiotics are in the pipeline, but for Gram negative organisms there is no antibiotic in the developing phase. This leaves us with only Colistin, Polymyxin B, and Tigicycline. However, Colistin and Polymyxin B are hazardous to use as they cause kidney and nerve damage and Tigecycline is not effective against Pseudomonas spp.
The present study was carried out to determine the distribution of bacterial pathogens causing nosocomial infections and their antibiogram.
| Materials and Methods|| |
Surveillance data from January to December 2011 was collected in Mohan Dai Oswal Hospital. A total of 1800 samples were taken of which maximum samples were urine (766) followed by blood (428) and pus (216), and so on.
Various samples were collected from the patients who were admitted for more than a week. All the samples were collected aseptically taking adequate precautions.
The samples were processed according to Clinical laboratory standards institute (CLSI)guidelines  and growth was subjected to sensitivity tests on Muller-hinton agar (MHA) plate according to the Kirby Bauer's method. Gram negative bacilli were isolated, identified, and subjected to antibiotic sensitivity tests. ESBL production was detected by using double disc diffusion method. A disc of ceftazidime and ceftazidime +clavulanic acid were placed at 20 mm distance (center to center). A difference in zones of inhibition of 5 mm or more was considered to be significant for ESBL producer.
| Results|| |
A total of 1800 samples from January to December 2011 were taken into account. It was found that the infection rate and percentage positivity (70%) was maximum in the pus samples. Distribution of various samples with their infection rates is shown in [Table 1].
Isolated organisms were further subjected to screening and confirmatory tests for ESBL. ESBL positivity was found to be considerably high in our hospital. It ranged from 82% to 90%. In the urine samples, ESBL positivity was 82% while in the pus samples it was about 90%.
ESBL positive strains were studied for sensitivity tests to all three generation of cephalosporins, combination formulations like tazobactum (Pipracillin+tazobactum), aminoglycosides, quinolones, carbapenems, tigecycline, Polymyxin B. Most of the organisms were resistant to all generation of cephalosporins and the combination formulations. It was also found that barring tigicycline and imipenem, the sensitivity to drugs like levofloxacin, moxifloxacin, amikacin, meropenem, polymyxin B, and pipracillin +tazobactum was very poor, which are frequently used to treat serious infections. However, among conventional drugs, Furadentin showed promising results. [Table 2] and [Table 3] show sensitivity pattern of ESBL positive Gram negative organisms in the urine and pus samples.
|Table 2: Sensitivity pattern of various antibiotics against ESBL positive Gram negative bacilli in urine sample|
Click here to view
|Table 3: Sensitivity pattern of various antibiotics against ESBL positive Gram negative bacilli in pus samples|
Click here to view
Almost similar results were obtained with all the samples.
| Discussion|| |
From this study, it is clear that nosocomial pathogens have shifted away from easily treatable bacteria to the more resistant and resilient types. As these organisms are prevalent in the hospital environment, they are also resistant to different kinds of antiseptics and disinfectants. This shift continues to present challenges for nosocomial infection control and prevention.
The underlying reason responsible for this shift is ESBL production by most of the Gram negative bacteria. Many factors as already mentioned lead to colonization of these organisms among the admitted patients and hospital staff. ,,
In our hospital, incidence of infection with ESBL producing organisms is between 80% and 90%. The main cause behind the high incidence is indiscriminate and injudicious use of cephalosporins and the last resort drugs - Carbapenems. Previous studies from India have reported that the prevalence of ESBL producers to be 6.6-91% [Table 4]. The wide variation in the prevalence is probably due to the variation in the risk factors and in the extent of antibiotic use. The prevalence of ESBL production is high in the referral centers and the ICUs where the patients are referred from the peripheral centers and where the high end antibiotic use is extensive. Studies, which were undertaken in Hubli, by Krishna et al.,  and, in New Delhi, by Wattal et al.,  revealed a markedly higher incidence of ESBL production, which can be attributed to the subjects being from the ICUs, where the prevalence and the risk factors, which are responsible for the emergence of the ESBL producers, are high. Other reasons for the high prevalence of the ESBL producers were indwelling catheters, endotracheal or nasogastric tubes, gastrostomies or tracheostomies, severity of the illness, the excessive use of cephalosporins, and a high rate of patient transfer from the peripheral centers. ,
Since our hospital is also a multispecialty hospital with patients in terminal stage of illness, critically ill, or referred from outside, the prevalence of ESBL was found to be high in our setup.
In conclusion, there is a high pervasiveness of ESBL producers among Enterobacteriaceae and the routine susceptibility tests, which are done, fail to detect the ESBL positive strains. The failure to detect these enzymes results in an uncontrolled spread of these organisms and finally, therapeutic failures. This study underscores the need for the routine detection of ESBL producers by specific tests.
The ESBL isolates were resistant to almost all of the third generation cephalopsrins tested. A similar high level of cephalosporin resistance was also noted by a study conducted by Rahal et al.  Wani et al.  reported a similarly higher percentage of resistance to Ceftazidime (99.2%), Cefotaxime (99.2%), and Ceftriaxone (99.5%). Jain et al.  reported resistance to Cefotaxime of more than 80.9% and up to 59.5% to Ceftazidime. Rafay et al.  observed 100% resistance to Cephalosporins - Ceftazidime, Cefotaxime, and Ceftriaxone.
Duttaroy and Mehta  showed resistance of 75% to Cefotaxime, 85% to Ceftazidime, and 60% to Ceftriaxone.
Hence, ESBL represent a class failure as far as use of cephalosporins is concerned. Besides, Cephalosporins may be ineffective even when routine microbiological methods report susceptibility (low level resistance. 
Among the quinolones, moxifloxacin was associated with more resistance than levofloixacin; Escherichia More Details coli isolates were totally resistant to moxifloxacin, while sensitivity to levofloxacin was also low. A similar high level of quinolone resistance has been reported elsewhere in India.  Resistance to fluoroquinolones is associated with ESBL production and is probably due to the same selection pressures.
Comparable resistance among the aminoglycosides like gentamicin and amikacin was seen akin to other reports. 
A noteworthy finding in our study was the almost 85% sensitivity to the fluoroquinolone Furadentin (Nitrofurantoin) among the urinary ESBL isolates belonging to the Enterobacteriacae group.
Fluoroquinolones constitute a reasonable choice for the treatment of urinary tract infections (UTIs) by ESBL-producing, susceptible organisms as evidenced in the study by Wani et al.,  who found 91.5% sensitivity among the urinary ESBL isolates. Low resistance of E. coli isolated from the urinary tract to this agent has been documented though susceptibility of ESBL isolates appear to be moderate in the study by Garau. 
Imipenem sensitivity among the ESBL isolates was found to be more than meropenem sensitivity especially in the urinary isolate. Regarded as the treatment of choice, success rates with carbapenems for ESBL producers consistently exceed 80%, and in no study has the outcome with carbapenems been surpassed.  Our findings are in agreement with this observation with high Imipenem sensitivity (100% Klebsiella, 77% Pseudomonas, 75% E. coli) in urinary isolates though it was lower for ESBl isolates in pus. The high imipenem sensitivity in the present study advocates the usage of carbapenem antibiotics as a therapeutic alternative in the wake of the increasing resistance rates, which were observed with the conventional β-lactam and non-β-lactam antibiotics. However, we need to keep in mind that the carbapenems are antimicrobials that are usually kept in reserve.  In the case of nonlife-threatening infections and in nonoutbreak situations, it is not necessary to administer carbapenems. This approach intends to preserve the therapeutic value of these precious drugs.
Our study highlights the role of Tigecycline in ESBL mediated MDR infections with Enterobacteriacae isolates showing 100% sensitivity. The first marketed member of glycylcyclines, Tigecycline is a 9-t-butylglycylamido derivative of minocycline. It evades common tetracycline efflux pumps and ribosomal protection mechanisms and has a bacteriostatic mode of activity. PubMed was searched for articles that evaluated the in vitro activity of tigecycline against Enterobacteriaceae with MDR or other clinically significant resistance patterns, as well as the clinical effectiveness of tigecycline against infections caused by such pathogens. We included 26 studies that evaluated the in vitro susceptibility to tigecycline of MDR Enterobacteriaceae (including ESBL-producing). These concluded that Tigecycline is microbiologically active against almost all of the ESBL or MDR E. coli isolates and the great majority of ESBL or MDR Klebsiella spp. isolates. 
Despite such encouraging results, Tigecycline use is beset with problems like no Pseudomonas spp. activity, blood concentration low for blood stream infections (BSI), reports of tigecycline-resistant Acinetobacter spp. (due to increased efflux).  Further evaluation of its clinical utility against such resistant Enterobacteriaceae, particularly regarding nonlabeled indications, is warranted.
Regarding the combination of tigecycline with other antibacterial agents, which may frequently be used in routine clinical practice for the treatment of severe infections, synergy studies have revealed an indifferent effect of most studied combinations against Gram-positive or Gram-negative bacteria. However, specific synergisms against certain Enterobacteriaceae have been noted, which might be worthy of further investigation. Specifically, time-kill experiments with Gram-negative pathogens confirmed synergism between tigecycline and ceftriaxone against K. pneumoniae, tigecycline and imipenem against Enterobacter cloacae, tigecycline and ceftazidime against M. morganii, tigecycline and trimethoprim/sulfamethoxazole against P. mirabilis and Serratia marcescens, as well as between tigecycline and amikacin against P. mirabilis and P. vulgaris. Moreover, antagonistic effects of tigecycline combinations have been observed only rarely. 
In our study, the polypeptide antibiotic Polymyxin B also demonstrated poor efficacy and large amount of antimicrobial resistance to these agents was noted, especially among the Enterobacteriacae. Some amount of sensitivity among the Pseudomonas isolates was observed (~50%).
Polypeptide antibiotics were discovered in the 1940s and have been in clinical use since the 1950s. They demonstrate bactericidal activity (destabilization of bacterial cell membrane) with a broad-spectrum of activity:
- Enterobacteriaceae (except Proteus, Providencia, Morganella, Serratia, Edwardsiella spp.)
- P. aeruginosa, Acinetobacter spp. !!! (not active against Burkholderia cepacia)
- Haemophilus influenza 
Despite this, there are only rare clinical reports for use against ESBL producing Enterobacteriaceae.  However, there are favorable effectiveness and safety profile against MDR A. baumannii and P. aeruginosa infections.  The main issue of concern in the use of this group is toxicity: Nephrotoxicity (20.2%) and neurotoxicity (7.36%) are the main reasons for restriction. 
| Conclusion|| |
The situation is quite dangerous. The time is not far when we will be back in the dark ages of the preantibiotic era. The need of the hour is to be alert of the gravity of this situation and take necessary measures to halt its progress.
Key issues in hospital infection control against ESBL pathogens
- Identification of colonized patients
- Most infected patients have previous colonization of the GI tract
- Culture of rectal swabs
- Use of selective media to isolate ESBL-producing Enterobacteriaceae
- Selective decontamination of the GI tract
- Limited value if there is coresistance to agents used in this regard
- Selective pressure for emergence of pathogens with advanced resistance patterns
- Failure to contain ESBL-producing organisms leads to heavier use of carbapenems and potential emergence of carbapenem-resistant pathogens
- Antibiotic restriction (3 rd generation cephalosporins)
- Has been shown to decrease the rate of isolation of ESBL-producing Enterobacteriaceae ,[ 29]
| References|| |
|1.||Greenwood D. Resistance to antimicrobial agents: A personal view. J Med Microbiol 1998;47:751-5. |
|2.||Rodriguez Bano J, Navarro MD, Romero L, Martinez- Martinez L, Muniain MA, Parea EJ, et al. Epidemiology and clinical features of infections caused by extended spectrum beta lactamase producing E.coli in non hospitalized patients. J Clin Microbiol 2004;42:1089-94. |
|3.||Peña C, Pujol M, Ricart A, Ardanuy C, Ayats J, Liñares J, et al. Risk factors for faecal carriage of Klebsiellapneumonie producing extended spectrum beta lactamase in the intensive care unit. J Hosp Infect 1997;35:9-16. |
|4.||Rice LB. Successful interventions for gram negative resistance to extended spectrum beta lactam antibiotics. Pharmacotherapy 1999;19:1205-85. |
|5.||Bradford PA, Cherubin CE, Idemyor V, Rasmussen BA, Bush K. Multiple resistant Klebsiella pneumonia strains from two Chicago Hospitals: Identification of the extended spectrum TEM-12 and TEM 10 Ceftazidime hydrolyzing beta lactamases in a single isolate. Antimicrob Agents Chemother 1994;38:761-6. |
|6.||Bradford PA, Urban C, Jaiswal A, Mariano N, Rasmussen BA, Projan SJ, et al. SHV-7, a novel cefotaximehydrolyzing beta lactamase identified in Escherichia coli isolates from hospitalized nursing home patients. Antimicrob Agents Chemother 1995;39:899-905. |
|7.||Yang Y, Bhachech N, Bradford PA, Jett BD, Sahm DF, Bush K. Ceftazidime resistant Klebsiella pneumonia and E. coli isolates producing TEM -10 and TEM 43 from St. Louis. Antimicrob Agents Chemother 1998;42:1671-6. |
|8.||Bradford PA, Urban C, Mariano N, Projan SJ, Rahal JJ, Bush K. Imipenam resistance in Klebsiella pneumonia is associated with the combination of ACT-1, a plasmid- mediated Amp C beta lactamase and the loss of an outer membrane protein. Antimicrob Agents Chemother 1997;41:563-9. |
|9.||Clinical and Laboratory Standards Institute. CLSI: Performance Standards for Antimicrobial Disk Susceptibility Tests; Approved Standard - 11th Ed. Clinical and Laboratory Standards Institute, 50 West Valley Road, Suite 2500, Wayne, PA 19087 USA M02-A1; January 2012; Vol 32; No 1. Available at http://antimicrobianos.com.ar/ATB/wp-content/uploads/2012/11/01-CLSI-M02-A11-2012.pdf [Last accessed on 2013 Aug 01]. |
|10.||Subha A, Ananthan S. Extended-Spectrum β-lactamase (ESBL) mediated resistance to third generation cephalosporins among Klebsiella pneumoniae in Chennai. Indian J Med Microbiol 2002;20:92-5. |
|11.||Krishna BV, Patil AB, Chandrasekhar MR. Extended-spectrum β-lactamase Producing Klebsiella pneumoniae in Neonatal Intensive Care Unit. Indian J Pediatr 2007;74:627-30. |
|12.||Jain A, Roy I, Gupta MK, Kumar M, Agarwal SK. Prevalence of extended-spectrum β-lactamase-producing Gram-negative bacteria in septicemic neonates in a tertiary care hospital. J Med Microbiol 2003;52:421-5. |
|13.||Wattal C, Sharma A, Oberoi JK, Datta S, Prasad KJ, Raveendr R. ESBL- An emerging threat to antimicrobial therapy. Microbiology Newsletter of Sir Ganga Ram Hospital, New Delhi; 2005;10:1-8. |
|14.||Rahal JJ, Urban C, Horn D, Freeman K, Segal-Maurer S, Maurer J, et al. Class restriction of cephalosporin use to control total cephalosporin resistance in nosocomial klebsiella. JAMA 1998;280:1233-7. |
|15.||Wani KA, Thakur MA, Siraj Fayaz A, Fomdia B, Gulnaz B, Maroof P. Extended Spectrum -Lactamase Mediated Resistance in Escherichia Coli in a Tertiary Care Hospital. Int J Health Sci (Qassim) 2009;3:155-63. |
|16.||Rafay AM, Al-Muharrmi Z, Toki R. Prevalence of extended spectrum -Lactamases producing isolates over a 1 year period at a University Hospital in Oman. Saudi Med J 2007;28:22-7. |
|17.||Duttaroy B, Mehta S. Extended spectrum -lactamases (ESBL) in clinical isolates of Klebsiella pneumoniae and Escherichia coli. Indian J Pathol Microbiol 2005;48:45-8. |
|18.||Paterson DL, Ko WC, Von Gottberg A, Casellas JM, Mulazimoglu L, Klugman KP, et al. Outcome of cephalosporin treatment for serious infections due to apparently susceptible organisms producing extended-spectrum beta-lactamases: Implications for the clinical microbiology laboratory. J Clin Microbiol 2001;39:2206-12. |
|19.||Garau J. Other antimicrobials of interest in the era of extended-spectrum beta-lactamases: Fosfomycin, nitrofurantoin and tigecycline. Clin Microbiol Infect 2008;14 Suppl 1:198-202. |
|20.||Paterson D L, Bonomo R A. Extended-Spectrum β-lactamase: A Clinical Update. Clin Microbiol Rev 2005; 18: 4: 657 - 686. |
|21.||Rodrigues C, Joshi P, Jani SH, Alphonse M, Radhakrishnan R, Mehta A. Detection of β-Lactamases in nosocomial Gram negative clinical isolates. Indian J Med Microbiol 2004;22:247-50. |
|22.||Kelesidis T, Karageorgopoulos DE, Kelesidis I, Falagas ME. Tigecycline for the treatment of multidrug-resistant Enterobacteriaceae: A systematic review of the evidence from microbiological and clinical studies. J Antimicrob Chemother 2008;62:895-904. |
|23.||Peleg AY, Adams J, Paterson DL. Tigecycline efflux as a mechanism for nonsusceptibility in Acinetobacter baumannii. Antimicrob Agents Chemother 2007;51:2065-9. |
|24.||Vouillamoz J, Moreillon P, Giddey M, Entenza JM. In vitro activities of tigecycline combined with other antimicrobials against multiresistant Gram-positive and Gram-negative pathogens. J Antimicrob Chemother 2008;61:371-4. |
|25.||Falagas ME, Bliziotis IA. Pandrug-resistant gram-negative bacteria: The dawn of the post-antibiotic era? Int J Antimicrob Agents 2007;29:630-6. |
|26.||Fleischer R, Boxwell D, Sherman KE. Nucleoside Analogues and Mitochondrial Toxicity. Clin Infect Dis 2004;38:e7-9. |
|27.||Falagas ME, Kasiakou SK. Colistin: The revival of polymyxins for the management of multidrug-resistant gram-negative bacterial infections. Clin Infect Dis 2005;40:1333-41. |
|28.||Koch-Weser J, Sidel VW, Federman EB, Kanarek P, Finer DC, Eaton AE. Adverse effects of sodium colistimethate. Manifestations and specific reaction rates during 317 courses of therapy. Ann Intern Med 1970;72:857-68. |
|29.||Kim JY, Sohn JW, Park DW, Yoon YK, Kim YM, Kim MJ. Control of extended-spectrum β-lactamase-producing Klebsiella pneumoniae using a computer-assisted management program to restrict third-generation cephalosporin use. J Antimicrob Chemother 2008;62:416-21. |
[Table 1], [Table 2], [Table 3], [Table 4]