Year : 2021 Month : February Volume : 8 Issue : 8 Page : 429-434.
Atit Dineshchandra Shah1, Urvashi Natubhai Limbachia2, Bhavin K. Prajapati3, Lata Patel4, Dharati Tusharbhai Shah5, Jayshri D. Pethani6, Nasiruddin M. Shaikh7, Hiral Shah8
1, 2, 3, 4, 5, 6, 7, 8 Department of Microbiology, Sardar Vallabhbhai Patel institute of Medical Sciences & Research Hospital, Smt. N.H.L. Municipal Medical College, Ahmedabad, Gujarat, India.
Dr. Atit Dineshchandra Shah,
Department of Microbiology,
Smt. N.H.L. Municipal Medical College,
Ahmedabad, Gujarat, India.
Email : firstname.lastname@example.org
Non-fermenting gram-negative bacilli (NFGNB) are a taxonomically diverse group of aerobic, non-spore forming bacilli that either do not use carbohydrates as a source of energy or degrade them through metabolic pathways other than fermentation.1 They are ubiquitous in nature, found as saprophytes inhabiting soil or water and some are also found as commensals in human and animal gut.2
In the hospital environment, they have been isolated from instruments such as ventilator machine, humidifiers, suction tubes, mattresses, other equipment and even from the skin of healthcare workers.3 They can cause device associated nosocomial infections and have the potential to spread from patient-to-patient via fomites or the hands of health care workers.4,5 These organisms are niche pathogens that primarily cause opportunistic healthcare-associated infections in patients who are critically ill or immunocompromised.5
NFGNB are innately resistant to many antibiotics and are known to acquire resistance by producing extended spectrum beta lactamase and metallobetalactamase.2 They pose a particular difficulty for the healthcare community because multidrug resistance is common and increasing among them and a number of strains have now been identified that exhibit resistance to all commonly used antibiotics. A variety of multidrug resistance makes treatment of infections caused by these pathogens both difficult and expensive.
NFGNB accounts for nearly 15 % of all gram-negative bacilli cultured from clinical specimens in a clinical microbiology laboratory.2,3 The important members of the group include Pseudomonas aeruginosa, Acinetobacter baumannii, Acinetobacter lwoffii, Stenotrophomonas maltophilia, Myroides spp. and Burkholderia cepacia. They cause various infections such as septicaemia, meningitis, pneumonia, urinary tract infections and surgical site infections (SSI).2
Identifying them in a routine microbiological laboratory was a diagnostic challenge as many of them are slow growers requiring battery of biochemical tests for their identification. This is not only time consuming and cumbersome, but many of them were also either misidentified or remained unidentified. Many of them were intrinsically resistant to various antibiotics. So, an automated system which identified them rapidly and accurately up to species level was indeed a need of an hour. Several automated systems were then developed and evaluated. Vitek-2 compact system is one such system developed by Biomerieux detects metabolic changes by fluorescence-based methods which facilitate the identification of gram-negative bacteria (GN cards) within 6 hours reducing turnaround time for identification. The instrument also processes the antimicrobial susceptibility cards (AST cards) until MIC’s are obtained. The system monitors the kinetics of bacterial growth and calculates minimum inhibitory concentrations (MIC) using a unique algorithm.6 So the current study was undertaken to isolate and identify NFGNB and to know their antibiotic susceptibility pattern by Vitek 2 method with their clinical significance.
The present observational study was conducted in department of Microbiology of a 1500 bed tertiary care teaching hospital, Ahmedabad during the period from March 2019 to February 2020. A total of 21053 samples were received for bacterial culture from OPD as well as admitted patients in different wards. Samples were plated on blood agar and MacConkey agar and incubated at 370 c. for 18 - 24 hours. The isolates that showed non lactose fermenting colonies on MacConkey agar or that do not grow on MacConkey agar were provisionally considered as non-fermenting gram-negative bacilli. Further identification was done on the basis of TSI changes and their identification as well as antimicrobial susceptibility testing was performed by Vitek 2 compact system using gram-negative identification card and antimicrobial susceptibility testing cards (GN ID and AST) (280 or 281). The cultures were incubated for 48 hours before declaring them as negative. To rule out chances of contamination, for specimens from non-sterile sites like urine and respiratory specimen like bronchoalveolar lavage (BAL) or endotracheal aspirate (ET), quantitative cultures were done and only those showing > 105 cfu / ml were considered for follow up. Whenever in doubt, a repeat sample was requested and only those showing growth of same organism in repeat culture were included in study.
Data was collected in a record form having primary details regarding specimen number, date of sample collection, date of reporting, specimen collection site and specimen type. The specimen record form has additional information whether specimen was positive or negative in culture, name of organisms isolated, colony count and their minimum inhibitory concentration (MIC) value in µg / ml and their final interpretation as sensitive (S), resistant (R) or intermediate (I) as per Clinical and Laboratory Standards Institute (CLSI) 2020 guidelines. Data so obtained as per case record form was presented in tabulated form as site wise distribution of clinical samples (Table 1 – whether OPD or indoor), specimen wise distribution of samples (Table 2), species distribution of NFGNB (Table 3) and their antimicrobial susceptibility pattern (Table 4). Data was further analysed using MS-Excel software.
Identification of NFGNB
The identification card (GN ID) contains substrate for various biochemical tests including tests for sugar assimilation, sugar fermentation, decarboxylase tests and other miscellaneous tests (urease, utilisation of malonate and tryptophan deaminase). Identification cards were inoculated with microorganism suspensions of 0.5 McFarland standards from a plate of pure culture using an integrated vacuum apparatus.
Antimicrobial Susceptibility Testing
Antimicrobial susceptibility testing with Vitek 2 compact system was performed using N281 card according to the manufacturer’s instructions. Antibiotics tested in AST N281 card included levofloxacin, gentamicin, cefepime, meropenem, imipenem, ticarcillin / clavulanic acid, doripenem, ceftazidime, cefoperazone / sulbactam, amikacin, ciprofloxacin, minocycline, tigecycline, colistin, trimethoprim / sulfamethoxazole (cotrimoxazole), cefotaxime, piperacillin / tazobactam, cefuroxime, ceftriaxone, tobramycin.
The Vitek-2 System automatically processes the antimicrobial susceptibility cards until MIC’s are obtained. The Vitek-2 compact system subsequently corrects MIC where necessary as per clinical category in accordance with the internal database of possible phenotypes for microorganism antimicrobial agent combinations.4
The Vitek 2 compact machine was validated using the standard strain as per manufacturer’s instructions. Pseudomonas aeruginosa American Type Culture Collection (ATCC) 27853 strain was used. During the study period, the control strain was checked at every 15 days.
A total of 21053 clinical samples received from in-patient and Out Patient Departments of SVP hospital, NHL Medical College, Ahmedabad during period from March 2019 to February 2020 were included in study. Majority of samples were received from indoor patients (95.01 %) including wards, emergency room (ER), intensive care unit (ICU) & critical care unit (CCU) and only 5.05 % (1065) samples received from OPD.
Majority of samples received were blood (34.5 %), urine (29.5 %), wound swabs (11.5 %), respiratory secretions (11.6 %) and fluids (5.6 %). A total of 6354 (30 %) of samples were positive in culture growing 6932 different isolates. Culture positivity was found maximum in endotracheal specimens (94 %) followed by wound swab (66 %) while lowest culture positivity was found in blood specimen (15 %). Some samples have grown more than one organism. Samples showing 2 or more isolates were repeated and only those isolates confirmed by repeated isolations were considered for study. Out of total of 6932 isolates, NFGNB were 2010 in number. (Isolation rate of 29 %). NFGNB were maximally isolated from respiratory specimen, endotracheal specimen, wound swabs and catheter tips. P. aeruginosa (52.7 %) and A. baumannii (36.5 %) were the commonest isolates followed by Stenotrophomonas maltophilia (2 %). Myroides spp. and A. lwoffii were isolated from 36 samples each (1.8 %). Other significant isolates were Burkholderia cepacia (N = 20, 1 %), Pseudomonas putida (N = 18, 0.9 %) and Sphingomonas paucimobilis (N = 11, 0.54 %). Acinetobacter junii was isolated from 7 samples while Elizabethkingia meningosepticum, P. fluorescens and Chryseobacterium indologenes were isolated in 5 samples. Other isolates were Achromobacter, Ochrobactrum anthropic, Ralstonia mannitolilytica, Delftia acidovorans, B. pseudomallei, & Oligella ureolytica.
Pseudomonas aeruginosa was the commonest isolate and was most susceptible to colistin (90.89 %) and least susceptible to ticarcillin / clavulanic acid (16.43 %). Carbapenem resistance was found in almost around 60 %. Amongst carbapenem, highest resistance was noted against meropenem (Sens - 38.68 %) followed by doripenem (Sens - 42.51 %). Imipenem was found to be most effective amongst carbapenem with sensitivity of 44.4 %. Piperacillin-tazobactam (Sens - 34.4 %) and aminoglycosides (Sens - 44 %) once used very frequently for pseudomonas infection were also found to be less sensitive. Resistance to ceftazidime (Sens - 41.74 %), cefepime (Sens - 46.29 %), ciprofloxacin (Sens - 38.75 %) and levofloxacin (Sens - 31.54 %) was also very high. Amongst fluoroquinolones, ciprofloxacin was found better than levofloxacin.
Acinetobacter spp. were most susceptible to colistin (Sens - 97.78 %) and tigecycline (Sens - 82.57 %). Minocycline is another drug showing good sensitivity against Acinetobacter baumannii (Sens - 53.4 %). Majority of other antibiotics showed high resistance of nearly 90 %. These were ceftazidime (Sens - 5.71 %), fluoroquinolones and aminoglycosides. Amongst fluoroquinolones, there was not much difference in susceptibility to ciprofloxacin (Sens - 6.36 %) and levofloxacin (Sens - 7.53 %). Carbapenem resistance was as high as 93 % for imipenem, meropenem and doripenem. Cefoperazone + sulbactam once thought to be active drug also showed poor susceptibility (Sens - 11 %) against A. baumannii.
In S. maltophilia, 34 strains out of 41 were susceptible to trimethoprim-sulfamethoxazole (Sens - 84 %) and 33 strains were susceptible to levofloxacin (Sens - 80.7 %). B. cepacia showed resistance to almost all drugs except minocycline (Sens - 64.3 %) and tigecycline (Sens - 57.1 %).
On observing the antimicrobial resistance pattern many isolates were seen to be resistant to 3 or more drugs. Multidrug resistance was very high amongst NFGNB. Pan drug resistance was also found in P. aeruginosa, A. baumannii and B. cepacia.
Isolation rate of around 15 % were found in most of studies while in our study it was 29 %. High isolation rate in our study was due to predominance of in-patient samples and a smaller number of OPD samples. Similar isolation rate of 31.62 % was found in study by Chang & Huang.7 Prevalence of NFGNB varies greatly from time to time and place to place. High isolation rate of NFGNB was found in similar studies carried out by Rao & Shivananda (66.8 %) and S. Sidhu & Pushpa Devi (45.9 %).8 Culture positivity was found maximum in endotracheal specimen (94 %) with highest isolation of NFGNB (52 %) from them followed by wound swab (37 %). NFGNB were predominantly isolated from pus or wound swab in many studies previously conducted in India.9,10 In our study, Pseudomonas aeruginosa (52.7 %) was commonest isolate followed by acinetobacter spp. (36.5 %), Stenotrophomonas maltophilia (2 %), Acinetobacter lwoffii (1.79 %), Myroids species (1.79 %) and Burkholderia cepacia (1 %). This is in concordance with many other studies by Benachinmardi KK et al., Martino R et al. & Gales AC et al.9,11,12
The rates of carbapenem resistance in non-glucose-fermenting gram-negative bacilli have been gradually increasing worldwide over the last 10 years and vary geographically.13,14 Recent studies from South Korea have reported the proportion of carbapenem resistance in acinetobacter to be as high as 32 to 56 % in hospitalized patients.15,16 In the United States, resistance rates have been reported from approximately 34 % to as high as 62.6 % (8 – 10). Reports from the National Healthcare Safety Network (NHSN) in the United States have demonstrated an increase from 33 % carbapenem resistance in 2006 to 2007 to 60 % among acinetobacter species isolates in 2009 to 2010. Carbapenem resistance in our study was as high as 93 % for acinetobacter species and 61 % for pseudomonas species. A study from Iran by Saderi H et al. also showed imipenem resistance of 68 % in Pseudomonas aeruginosa from hospitalised burn patients.17 A study from India by Agarwal S et al. also showed imipenem resistance of 90.54 % in Acinetobacter baumannii and 52 % in Pseudomonas aeruginosa.18 Similar results of high carbapenem resistance was also observed in a study conducted by Sharma D et al. in Jaipur, India.19
P. aeruginosa also showed poor sensitivity to cephalosporins-ceftazidime (41.7 %) and cefepime (46.3 %), fluoroquinolones-ciprofloxacin (38.8 %) and levofloxacin (31.5 %) and aminoglycosides–gentamycin (43.8 %) and amikacin (44.4 %), while for A. baumannii resistance in these groups were very high compared to P. aeruginosa. For A. baumannii, sensitivity to ceftazidime was (5.7 %), cefepime (6.9 %), ciprofloxacin (6.4 %), levofloxacin (7.5 %) and gentamycin (9.3 %). Resistance in these pathogens may arise due to intrinsic mechanisms or may be acquired through mutations or plasmids. Sometimes resistance may develop during prolonged therapy, which was initially effective. A variety of resistance mechanisms have been identified including enzyme production (e.g., β-lactamases, AmpC, carbapenemase), overexpression of efflux pumps (fluoroquinolones), porin deficiencies (Beta-lactam antibiotics) and target-site alterations (aminoglycosides and fluoroquinolones). In some cases, these resistance mechanisms affect the susceptibility of individual antibiotics differently (even in the same group); this is the reason why some isolates may be resistant to meropenem, but not imipenem, or resistant to amikacin, but not tobramycin. Results similar to our study were also observed by studies carried out in same hospital previously by Parimal Patel et al.20 in 2013.
S. maltophilia infections are also a serious concern, as this microorganism is intrinsically resistant to a wide range of antimicrobial drugs and data on clinical effectiveness is only available for sulfamethoxazole / trimethoprim and fluoroquinolones. In our study we have found good susceptibility to both sulfamethoxazole / trimethoprim (84 %) and fluoroquinolones–levofloxacin (80.8 %). Results similar to our study were obtained by Sun E et al.21 from China.
Colistin resistance was observed 9 % in Pseudomonas aeruginosa and 2.2 % in A. baumannii
Pseudomonas aeruginosa and acinetobacter species were commonest NFGNB isolated in our study. P. aeruginosa showed high degree of resistance to beta-lactam, beta-lactam-beta-lactamase inhibitor combinations, fluoroquinolones, aminoglycosides and carbapenems. Colistin remained drug of choice for both isolates. A. baumannii had shown good susceptibility to minocycline and tigecycline too. For other NFGNB like B. cepacia and myroides species, minocycline is the drug of choice, showing good susceptibility. Resistant pattern among nosocomial bacterial pathogens may vary from country to country and from region to region. Different species of NFGNB have shown a varied sensitivity pattern in our study. Therefore, identification of NFGNB and monitoring their susceptibility pattern are important for proper management of these infections. Treatment of infections by NFGNB presents a therapeutic challenge to clinicians due to their increasing levels of resistance to several classes of antibiotics. Early diagnosis and institution of empirical antibiotic therapy based on recent antibiogram of institute would reduce mortality and improve patient management. The Vitek 2 compact system identifies NFGNB along with their antibiotic susceptibility pattern within a time period of 8 to 16 hours helping in early institution of therapy, thereby reducing mortality and providing better care and management of patients. Implementation of stringent antibiotic stewardship and strict infection control practices will be required to prevent the emergence or slow down the spread of multi drug and pan drug resistance strains of NFGNB.
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Financial or other competing interests: None.
Disclosure forms provided by the authors are available with the full text of this article at jebmh.com.
Disclosure forms provided by the authors are available with the full text of this article at jebmh.com
Financial or other competing interests: None.
Copyright © 2021 Atit Dineshchandra Shah et al. This is an open access article distributed under Creative Commons Attribution License [Attribution 4.0 International (CC BY 4.0)]
Shah AD, Limbachia UN, Prajapati BK, et al. Occurrence of non-fermenting Gram-negative bacilli and their in vitro susceptibility pattern by Vitek 2 at a tertiary care teaching hospital – an observational study. J Evid Based Med Healthc 2021;8(08):429-434. DOI: 10.18410/jebmh/2021/84