|Year : 2016 | Volume
| Issue : 2 | Page : 271-275
Presence of metallo-beta-lactamases (MBL), extended-spectrum beta-lactamase (ESBL) & AmpC positive non-fermenting Gram-negative bacilli among Intensive Care Unit patients with special reference to molecular detection of blaCTX-M & blaAmpC genes
Richa Gupta1, Abida Malik1, Meher Rizvi1, Moied Ahmed2
1 Department of Microbiology, J. N. Medical College, Aligarh Muslim University, Aligarh, India
2 Department of Anaesthesiology, J. N. Medical College, Aligarh Muslim University, Aligarh, India
|Date of Submission||02-Apr-2014|
|Date of Web Publication||1-Dec-2016|
Department of Microbiology, J. N. Medical College, Aligarh Muslim University, Aligarh 202 002, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background & objectives: Non-fermenting Gram-negative bacilli (NFGNB) including Pseudomonas aeruginosa and Acinetobacter baumannii have been implicated in a variety of infections, particularly in the Intensive Care Units (ICUs). This study was aimed to overview the burden of multidrug-resistant NFGNB causing infections in ICU and also to assess the occurrence of extended-spectrum beta-lactamases (ESBLs), AmpC and metallo-beta-lactamases (MBLs) among these isolates.
Methods: Bacterial culture, identification and antibiotic susceptibility were carried out. ESBLs and AmpC were detected both phenotypically and genotypically. MBL was detected by modified Hodge and imipenem-ethylenediaminetetraacetic acid double-disc synergy test.
Results: NFGNB represented 45 (37%) of total 121 Gram negative isolates. Multidrug resistance was observed in 66.9 per cent and 72.5 per cent isolates of P. aeruginosa and A. baumannii, respectively. Detection by phenotypic methods showed presence of ESBL, AmpC and MBL in 21.4, 51.1 and 21.4 per cent isolates, respectively. When detected genotypically by polymerase chain reaction, ESBL and AmpC were detected in 21.4 and 41.4 per cent of NFGNB isolates, respectively. BlaCTX-M (21.4%) was the most prevalent gene responsible for ESBL production.
Interpretation & conclusions: Most of the NFGNB isolated from ICU patients were multidrug-resistant and producers of ESBL, AmpC and MBL. A regular surveillance is required to detect ESBL, AmpC and MBL producers, especially in ICU patients.
Keywords: Acinetobacter baumannii - Intensive Care Unit - multidrug-resistance - Pseudomonas aeruginosa
|How to cite this article:|
Gupta R, Malik A, Rizvi M, Ahmed M. Presence of metallo-beta-lactamases (MBL), extended-spectrum beta-lactamase (ESBL) & AmpC positive non-fermenting Gram-negative bacilli among Intensive Care Unit patients with special reference to molecular detection of blaCTX-M & blaAmpC genes. Indian J Med Res 2016;144:271-5
|How to cite this URL:|
Gupta R, Malik A, Rizvi M, Ahmed M. Presence of metallo-beta-lactamases (MBL), extended-spectrum beta-lactamase (ESBL) & AmpC positive non-fermenting Gram-negative bacilli among Intensive Care Unit patients with special reference to molecular detection of blaCTX-M & blaAmpC genes. Indian J Med Res [serial online] 2016 [cited 2020 Jun 1];144:271-5. Available from: http://www.ijmr.org.in/text.asp?2016/144/2/271/195043
Non-fermenting Gram-negative bacilli (NFGNB) including Pseudomonas aeruginosa and Acinetobacter baumannii have been implicated in a variety of infections, including bacteraemia, urinary tract and surgical site infections among patients admitted in Intensive Care Unit (ICU) , . These may be intrinsically resistant or may have acquired resistance to antibiotics due to impermeability of the cell surface, multidrug efflux pumps and production of ß-lactamases [AmpC ß-lactamase, extended-spectrum ß-lactamases (ESBLs) and metallo-beta-lactamases (MBLs)]  . Multiple beta-lactamase-producing P. aeruginosa can cause major therapeutic failure and poses a significant clinical challenge. Reports on carbapenemase-producing NFGNB are on the rise globally due to the increased carbapenem usage and selection of resistant bacteria under antibiotic pressure  . Therefore, early identification and detection of isolates that produce these enzymes are essential to avoid therapeutic failures and nosocomial outbreaks.
This study was designed to assess the burden of multidrug-resistant P. aeruginosa and A. baumannii in ICU patients. The occurrence of ESBL, AmpC and MBL among these isolates was also assessed.
| Material & Methods|| |
The present study was carried out in the department of Microbiology on patients admitted to the ICU of J. N. Medical College, Aligarh Muslim University, Aligarh, India from February 2012 to October 2013. Totally, 125 patients admitted to the ICU were included in the study. A complete history was taken from each patient. Informed written consent was obtained before the study from all the patients, and the study was performed after getting approval from the Institutional Ethics Committee.
The patients were chosen consecutively, and clinical samples were obtained from each patient (endotracheal aspirate, blood, pus, urine). All specimens were collected aseptically and were promptly sent to the microbiology laboratory. All samples were collected within 48 h of the patient admission in the ICU and those collected after 48 h of admission were not included in the study. Wounds (surgical site infections) have been classified according to the Southampton grading  . The majority of the cases belonged to Grade IV (purulent discharge along the wound) and Grade V (wound dehiscence). Standard methods for isolation and identification of NFGNB  were used.
Susceptibility testing of bacterial isolates was performed using the disc diffusion method as described by the Clinical and Laboratory Standards Institute  . Antimicrobial discs used were imipenem (10 µg), cefpodoxime (10 µg), cefotaxime (30 µg), cefepime (30 µg), cefixime (5 µg), cefoperazone (75 µg), cefoperazone/sulbactam (75/10 µg), ticarcillin (75 µg), piperacillin (100 µg), piperacillin/tazobactam (100/10 µg), ceftazidime (30 µg), ceftazidime/clavulanic acid (30/10 µg), cefotaxime/clavulanic acid (30/10 µg), ceftriaxone (30 µg), amikacin (30 µg), gentamicin (10 µg), tobramycin (10 µg), ofloxacin (5 µg), levofloxacin (5 µg), polymixin B (300 units) and colistin (10 µg). All discs were obtained from Hi-Media Labs, Mumbai, India.
Phenotypic methods for ESBL detection: NFGNB isolates were first screened for the production of ESBL by the disc diffusion method (screening test) using cefotaxime, ceftriaxone, cefepime and ceftazidime  and later on confirmed by the cephalosporin/clavulanate combination disc (disc potentiation test)  and double-disc synergy test , . Escherichia coli ATCC 25922 (non-ESBL producer) was used as a control strain.
Phenotypic methods for AmpC detection: Cefoxitin discs were used to screen AmpC producers, by disc diffusion method  . Isolates resistant to cefoxitin were considered as potential AmpC producers.
Phenotypic methods for MBL detection: The isolates were tested for sensitivity to imipenem (10 μg) using Kirby-Bauer method as recommended by the CLSI  . All the isolates with a zone of inhibition ≤16 mm or which demonstrated heaping, or if the zone was >16 but ≤20 mm, were tested for MBL production; however, there is no CLSI guideline for MBL detection available for P. aeruginosa. These isolates were confirmed by modified Hodge test and imipenem-ethylenediaminetetraacetic acid (EDTA) double-disc synergy test , .
Genotypic methods for the detection of ESBL and AmpC production: Template DNA was prepared from freshly cultured bacterial isolates by suspending bacterial colonies in 50 µl of molecular grade water and then heating at 95°C for five minutes and immediately chilling at 4°C. Molecular detection of blaCTX-M , blaTEM , blaSHV and blaAmpC was performed using polymerase chain reaction (PCR) according to methods described previously with minor modifications (thermal profile of blaCTX-M  , primer profiles of blaCTX-M , blaTEM , blaSHV , blaAmpC  ). The primers and cycling conditions for detection of blaAmpC genes were the same as those described by Shahid et al and Féria et al . The quality-control strain, Klebsiella pneumoniae ATCC 700603 (ESBL producer) was used.
| Results & Discussion|| |
Among the 125 patients admitted to the ICU, 160 isolates were identified. Of these, Gram-negative bacilli, 121 (75.6%) predominated, followed by 22 (13.8%) Gram-positive cocci and 10.6 per cent (n=17) fungal isolates. NFGNB represented 45 (37%) of the Gram-negative isolates (n=121) of which P. aeruginosa (n=35, 29%) was the incriminatory pathogen in majority, followed by A. baumannii (n=10, 8%).
Antimicrobial resistance was observed to be higher in A. baumannii than in P. aeruginosa. Antibiotic resistance pattern of P. aeruginosa and A. baumannii is shown in [Table 1]. [Table 2] depicts the organisms isolated from different samples of patients in ICU. The positivity for ESBL, AmpC and MBL by phenotypic methods is shown in [Table 3].
|Table 1. Antibiotic resistance pattern of 45 non-fermenting Gram-negative bacilli detected by disc diffusion method |
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|Table 2. Distribution of pathogens isolated from endotracheal aspirate and urinary tract infection in Intensive Care Unit patients |
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|Table 3. Number and percentage of extended-spectrum β-lactamase (ESBL)-producing Pseudomonas aeruginosa and Acinetobacter baumannii by phenotypic methods |
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BlaCTX-M , blaSHV , blaTEM and blaAmpC genes were detected in phenotypic ESBL and AmpC producers. BlaCTX-M was the predominant gene among ESBL producers as it was observed in four (11.4%) isolates of P. aeruginosa and one (10%) A. baumannii. BlaAmpC was detected in 15 (42.8%) P. aeruginosa and four (40%) A. baumannii isolates. BlaSHV and blaTEM were not detected in any of the isolates.
NFGNB including P. aeruginosa and A. baumannii have been implicated in a variety of ICU infections. In this study, P. aeruginosa represented 29 per cent of isolates; similar results were reported by Hadadi et al . In other studies, P. aeruginosa represented 15.6 per cent of the total isolates , . A. baumannii represented eight per cent of Gram-negative ICU infections in this study. However, other investigators found a higher incidence of A. baumannii (20.5 and 24.1%, respectively) , .
In this study, lower respiratory tract infections (LRTIs) were the most common infection in ICU patients (34.3%). Al-Ghamdi et al reported 8.9 per cent LRTIs among ICU cases. In our study, P. aeruginosa and A. baumannii were frequently isolated from LRTIs (40 and 7.3%, respectively), as also shown by Abd El-Fattah  .
In our study 76.1 and 70.2 per cent of NFGNB were resistant to fluoroquinolones and aminoglycosides, respectively. Antibiotic resistance is a serious problem in developing countries, especially due to the easy availability of antibiotics over the counter , . The resistance of P. aeruginosa and A. baumannii to ceftazidime (68.5%, 70%), cefotaxime (57.1%, 70%) and cefpodoxime (65.7%, 70%) was comparable to results reported by others , .
Screening by disc diffusion method in this study revealed that 24.3 per cent of NFGNB were ESBL producers. These results were comparable to that of Aggarwal et al , however, lower than results reported by Jiang et al . Confirmation with double-disc synergy test (DDST) and disc combination tests revealed that 18.5 per cent of NF Gram-negative isolates were ESBL producers as also reported by Jiang et al . For AmpC production, both disc diffusion test and screening by cefoxitin disc revealed same results that 51.4 per cent of P. aeruginosa and 50 per cent of A. baumannii isolates were AmpC producers. Similar results were reported by Bhattacharjee et al  . The most prevalent gene for ESBL production was blaCTX-M which was detected in 11.4 per cent of Pseudomonas isolates, while blaTEM and blaSHV genes were not detected in any of the isolates. These results were in agreement with Picão and Gales  . PCR detected blaAmpC gene in 42.8 per cent of P. aeruginosa and 40 per cent of A. baumannii isolates. These results were comparable to that of Khanal et al .
Comparison between modified Hodge test and DDST in our study revealed that DDST was more sensitive for detecting MBL. The same observation was reported by Jesudason et al . In this study, amikacin, tobramycin, imipenem, polymyxin B and colistin demonstrated maximum sensitivity against NFGNB. Therefore, use of these antibiotics should be restricted to severe infections, especially in critically ill ICU patients, to avoid rapid emergence of resistant strains.
In conclusion, our results showed isolation of a high percentage of NFGNB in ICU patients' samples, which were multidrug resistant and producers of ESBL, AmpC and MBL. A regular surveillance of antimicrobial susceptibility status of such isolates is necessary to curb the infection.
Conflicts of Interest: None.
| References|| |
Nseir S, Di Pompeo C, Brisson H, Dewavrin F, Tissier S, Diarra M, et al.
Intensive care unit-acquired Stenotrophomonas maltophilia
: incidence, risk factors, and outcome. Crit Care
Malini A, Deepa E, Gokul BN. Nonfermenting Gram-negative bacilli infections in a tertiary care hospital in Kolar, Karnataka. J Lab Physicians
Aloush V, Navon-Venezia S, Seigman-Igra Y, Cabili S, Carmeli Y. Multidrug-resistant Pseudomonas aeruginosa
: risk factors and clinical impact. Antimicrob Agents Chemother
Gales AC, Jones RN, Forward KR, Liñares J, Sader HS, Verhoef J. Emerging importance of multidrug-resistant Acinetobacter
species and Stenotrophomonas maltophilia
as pathogens in seriously ill patients: geographic patterns, epidemiological features, and trends in the SENTRY Antimicrobial Surveillance Program (1997-1999). Clin Infect Dis
(Suppl 2): S104-13.
Wilson APR, Gibbons C, Reeves BC, Hodgson B, Liu M, Plummer D. Surgical wound infection as a performance indicator: agreement of common definitions of wound infection in 4773 patients. BMJ
Collee JG, Marr W. Culture of bacteria. In: Fraser AG, Collee JG, Marmion BP, Simmons A, editors. Mackie and McCartney practical microbiology
. 14 th
ed. London: Churchill Livingstone; 1996. p. 113-30.
Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial susceptibility testing: Seventeenth informational supplement.
M100-S17. Wayne, PA: CLSI; 2007.
Paterson DL, Bonomo RA. Extended-spectrum beta-lactamases: a clinical update. Clin Microbiol Rev
Jarlier V, Nicolas MH, Fournier G, Philippon A. Extended broad-spectrum beta-lactamases conferring transferable resistance to newer beta-lactam agents in Enterobacteriaceae
: hospital prevalence and susceptibility patterns. Rev Infect Dis
Lee K, Lim JB, Yum JH, Yong D, Chong Y, Kim JM, et al. bla
(VIM-2) cassette-containing novel integrons in metallo-beta-lactamase-producing Pseudomonas aeruginosa
and Pseudomonas putida
isolates disseminated in a Korean hospital. Antimicrob Agents Chemother
Shahid M, Malik A, Agrawal M, Singhal S. Phenotypic detection of extended-spectrum and AmpC beta-lactamases by a new spot-inoculation method and modified three-dimensional extract test: comparison with the conventional three-dimensional extract test. J Antimicrob Chemother
Shahid M, Ensor VM, Hawkey PM. Emergence and dissemination of Enterobacteriaceae
with plasmid-mediated CMY-6 and CTX-M-15 beta-lactamases in a community in North-India. World J Microbiol Biotechnol
Féria C, Ferreira E, Correia JD, Gonçalves J, Caniça M. Patterns and mechanisms of resistance to beta-lactams and beta-lactamase inhibitors in uropathogenic Escherichia coli
isolated from dogs in Portugal. J Antimicrob Chemother
Hadadi A, Rasoulinejad M, Maleki Z, Yonesian M, Shirani A, Kourorian Z. Antimicrobial resistance pattern of gram-negative bacilli of nosocomial origin at 2 university hospitals in Iran. Diagn Microbiol Infect Dis
Chim H, Tan BH, Song C. Five-year review of infections in a burn intensive care unit: high incidence of Acinetobacter baumannii
in a tropical climate. Burns
Izquierdo-Cubas F, Zambrano A, Frómeta I, Gutiérrez A, Bastanzuri M, Guanche H, et al.
National prevalence of nosocomial infections. Cuba 2004. J Hosp Infect
Al-Ghamdi S, Gedebou M, Bilal NE. Nosocomial infections and misuse of antibiotics in a provincial community hospital, Saudi Arabia. J Hosp Infect
Abd El-Fattah SM. Evaluation of antibiotic resistance among Gram-ve bacilli isolated from critically ill patients: Relation to risk factors and liberal use of antibiotics. M.Sc Thesis in Medical Microbiology and Immunology. Faculty of Medicine. Giza, Egypt: Cairo University; 2008.
Giamarellou H, Antoniadou A, Kanellakopoulou K. Acinetobacter baumannii:
a universal threat to public health? J Hosp Infect
Martínez JL, Baquero F. Interactions among strategies associated with bacterial infection: pathogenicity, epidemicity, and antibiotic resistance. Clin Microbiol Rev
McGowan JE Jr. Resistance in nonfermenting gram-negative bacteria: multidrug resistance to the maximum. Am J Med
(Suppl 1). S29-36.
Aggarwal R, Chaudhary U, Bala K. Detection of extended-spectrum beta-lactamase in Pseudomonas aeruginosa
. Indian J Pathol Microbiol
Jiang X, Zhang Z, Li M, Zhou D, Ruan F, Lu Y. Detection of extended-spectrum beta-lactamases in clinical isolates of Pseudomonas aeruginosa
. Antimicrob Agents Chemother
Bhattacharjee A, Anupurba S, Guar A, Sen MR. The prevalence of inducible AmpC ß-lactamase producing Pseudomonas aeruginosa
in a tertiary care hospital in northern India. Indian J Med Microbial
Picão RC, Gales AC. Extended-spectrum beta-lactamase-producing (ESBL) Pseudomonas aeruginosa:
nightmare or imagination? Prát Hosp
Khanal S, Joshi DR, Bhatta DR, Devkota U, Pokhrel BM. ß-lactamase-producing multidrug-resistant bacterial pathogens from tracheal aspirates of intensive care unit patients at national institute of neurological and allied sciences, Nepal. ISRN Microbiol
Jesudason MV, Kandathil AJ, Balaji V. Comparison of two methods to detect carbapenemase & metallo-beta- lactamase production in clinical isolates. Indian J Med Res
[Table 1], [Table 2], [Table 3]