| Abstract|| |
Background: Citrobacter is an important nosocomial pathogen and its multi-drug resistant (MDR) isolates are increasingly being reported across the globe. They are known to produce extended spectrum beta lactamase (ESBL) and harbor CTX-M gene. Objective: The aim was to isolate Citrobacter sp. from clinical specimens, analyze their MDR status and look for the presence of CTX-M gene. Materials and Methods: In this prospective study, Citrobacter isolates positive for ESBL on screening, were confirmed by combined disc method along with minimum inhibitory concentration (MIC) for cefotaxime. In selected cefotaxime resistant isolates, multiplex polymerase chain reaction was done for blaCTX-M gene. Results: Of 146 Citrobacter sp. isolated, most (73%) were from admitted patients and hospital stay of >72 h and prior antibiotic intake were the most common associated factors. Maximum isolates were from pus (41.1%). Citrobacter freundii was the commonest species (49%) followed by Citrobacter koseri (28%); 79 were ESBL producers. Seventy were cefotaxime resistant as shown by MIC. blaCTX-M gene was detected in 15/40 of these isolates, all belonged to CTX-M group 1. Conclusion: Overall incidence of Citrobacter in our setup is low, but they were mostly MDR, and ESBL production was high, which is a cause of concern. blaCTX-M gene detection is important because of its rapid transmission to other bacterial species.
Keywords: Extended spectrum beta lactamases, minimum inhibitory concentration, nosocomial infection
|How to cite this article:|
Mohan S, Agarwal J, Srivastava R, Singh M. Observations on Citrobacter species from a tertiary care health center with special reference to multi-drug resistance and presence of CTX-M gene. Indian J Pathol Microbiol 2014;57:439-41
|How to cite this URL:|
Mohan S, Agarwal J, Srivastava R, Singh M. Observations on Citrobacter species from a tertiary care health center with special reference to multi-drug resistance and presence of CTX-M gene. Indian J Pathol Microbiol [serial online] 2014 [cited 2021 Oct 16];57:439-41. Available from: https://www.ijpmonline.org/text.asp?2014/57/3/439/138746
| Introduction|| |
Infections with multi-drug resistant (MDR) Citrobacter sp. have been increasing across the globe and thus present a challenge for the clinician and clinical microbiologist because of their association with nosocomial infection. The commonly implicated species are Citrobacter freundii and Citrobacter koseri., They are associated with respiratory infections, urinary tract infections, intra-abdominal infections, meningitis and hospital acquired bacteremia. Organisms producing extended spectrum beta lactamases (ESBLs) are important clinically and are a cause of concern mainly due to failure of therapy with cephalosporins. In recent years, CTX-M ESBLs are being frequently reported from various countries. CTX-M was first discovered in 1989 and unlike other ESBLs, CTX-M family constitute a complex and nonhomogeneous group of enzymes.  Its lineage is differentiated into at least five main clusters. It is reported to have a tendency for rapid dissemination among other bacteria as it is associated with mobile elements such as ISEcp1. , Numerous international studies have described the occurrence of various CTX-M types in Citrobacter, but only scanty data is available from India. This study provides information regarding MDR status and presence of CTX-M gene in Citrobacter sp. isolated from clinical specimens at a tertiary care health center.
| Materials and methods|| |
Study site and subjects
It was a prospective observational study and all clinical specimens from all age groups, referred to the Microbiology Department of a tertiary care hospital for culture and antibiotic sensitivity, from May 2012 to April 2013 were included. Various specimens were blood, cerebrospinal fluid, urine, pus, swabs and body fluids and so on. Bacterial isolates were identified using standard conventional methods. 
Antibiotic sensitivity testing
Antibiotic susceptibility of isolates was done on Muller Hinton agar using Kirby Bauer method (Clinical and Laboratory Standards Institute [CLSI] 2013).  Antibiotic discs were obtained from HiMedia, Mumbai, India. Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 25923 were used as controls. MDR was defined as resistance for >3 class of antibiotics. 
Minimum inhibitory concentration
Minimum inhibitory concentration (MIC) of cefotaxime (Laczene Biosciences, Mumbai) was done by agar dilution method for MDR Citrobacter isolates. Doubling dilutions of cefotaxime powder from 0.5 μg/ml to 16 μg/ml were used and MIC of ≥4 μg/ml was interpreted as resistant (CLSI 2013).  Escherichia coli ATCC 29522 was used as control.
Phenotypic test for extended spectrum beta lactamase detection
Confirmatory phenotypic test (combined disc diffusion test; CLSI 2013)  was done for strains resistant to cefotaxime disk on disc diffusion test.
Detection of CTX-M genes
The presence of CTX-M genes was detected by polymerase chain reaction (PCR) amplification using primers specific for blaCTX-M alleles 1, 2, 8, 9, and 25 in selected isolates. 
| Results|| |
During the study period, 146 Citrobacter (nonduplicate) were obtained. Pus was the commonest sample from where Citrobacter were isolated (41.1%). Of the 146 Citrobacter isolates, 34 (23%) were from outpatient and remaining 112 (7%) were from admitted patients, namely from the general surgery ward (46.9%) and Intensive Care Unit (ICU) (30.4%). Most common species identified were C. freundii (49%) and C. koseri (28%) followed by C. farmeri (8.2%), C. braakii (5.4%), C. werkmanii (5.4%) and C. Gilleni (3.4%). Hospital stay of >72 h at the time of sample collection was the most common finding, present in nearly three quarters of the admitted patients. Other factors present were co-morbidity (diabetes, malignancy, hypertension etc.), invasive devices and surgery in the recent past. Over 50% of patients with Citrobacter infection, gave a history of penicillin and cephalosporins consumption in last 7-10 days. Among the antibiotics tested for susceptibility, carbapenem emerged as the most sensitive class of antibiotic, followed by penicillin group [Table 1]. In this study, 55.4% (81/146) of isolates were MDR and they were mostly from neurosurgery ward (48.1%). Of 130 isolates which were cefotaxime resistant as detected by disc diffusion test only 107 isolates had MIC >4 μg/ml that is, resistant; of these resistant isolates, 36 had MIC >8 μg/ml and 44 had MIC of >16 μg/ml. Seventy-nine of these 107 cefotaxime resistant isolates (73.8%) were confirmed ESBL producers by combined disc method. Presence of blaCTX-M was determined in 40 randomly selected cefotaxime resistant ESBL confirmed Citrobacter isolates and 15 (37.5%) tested positive all belonging to CTX-M group 1.
| Discussion|| |
Microorganisms belonging to genera Citrobacter sp. rarely cause infection in normal hosts. The importance lies in the fact that they tend to produce serious nosocomial outbreaks and that they are increasingly becoming resistant to antibiotic.  Very few studies in India have dealt with Citrobacter and its antibiotic sensitivity pattern.  In this study, majority of the Citrobacter isolates were recovered from pus samples (41.1%) and Citobacter freundi (48%) was the most common species, followed by C. koseri (28%), similar to findings by Khanna et al. and Samonis et al. , While few others have reported C. koseri to be the predominant species. ,, We also isolated rarer species like C. farmeri (8.2%), C. braakii (5.4%), C. werkmanii (5.4%) and C. Gilleni (3.4%) not reported in other studies. These are shown to be potential pathogens.  Maximum number of isolates were received from surgical ward (46.9%) followed by ICU (30.4%) similar to a previous study.  In this study, majority patients had a history of hospital stay of >72 h. Duration of stay in the hospital, ICU stay, previous hospital admissions in last 1-year and recent exposure to antibiotics have been reported to be the common risk factors associated with Citrobacter infection. 
Extended spectrum beta lactamase production was found in 54% (79/146) isolates, which was similar with the finding of Khanna et al.  (57%) and Rizvi et al.  (62%), but much lower than that reported by some other studies. , Indiscriminate and overuse of extended spectrum antibiotics is shown to be the common reason for high prevalence of MDR and ESBL in hospital setup. , In this study, we looked for the presence of CTX-M beta-lactamases by multiplex PCR. 37.5% of tested isolates harbored blaCTX-M and all belonged to genogroup 1, although this is much lower than that previously reported from North India.  blaCTX-M gene rapidly disseminate and are now frequently being reported from countries all over the world. In our study, though overall incidence of infection by Citrobacter was low; they were mostly MDR isolates. Our study reports the presence of CTX-M in Citrobacter sp. which is important for epidemiological purpose because of its tendency for rapid dissemination to unrelated bacterial species.  Proper aseptic and barrier precaution along with appropriate antibiotic policy are needed to prevent dissemination of such resistant strains in hospital setup.
| References|| |
Khanna A, Singh N, Aggarwal A, Khanna M. The antibiotic resistance pattern in Citrobacter
species: An emerging nosocomial pathogen in a tertiary care hospital. J Clin Diagn Res 2012;6:
Samonis G, Karageorgopoulos DE, Kofteridis DP, Matthaiou DK, Sidiropoulou V, Maraki S, et al. Citrobacter
infections in a general hospital: Characteristics and outcomes. Eur J Clin Microbiol Infect Dis 2009;28:61-8.
Bauernfeind A, Grimm H, Schweighart S. A new plasmidic cefotaximase in a clinical isolate of Escherichia coli
. Infection 1990;18:294-8.
Cantón R, González-Alba JM, Galán JC. CTX-M Enzymes: Origin and diffusion. Front Microbiol 2012;3:110.
Karim A, Poirel L, Nagarajan S, Nordmann P. Plasmid-mediated extended-spectrum beta-lactamase (CTX-M-3 like) from India and gene association with insertion sequence ISEcp1. FEMS Microbiol Lett 2001;201:237-41.
Collee JG, Miles RS, Watt B. Identification of bacteria. In: Collee JG, Fraser AG, Marmion BP, Simmons A, editors. Practical Medical Microbiology. 14 th
ed., vol. 2. Singapore: Churchill Livingstone Publishers, Longman; 2003. p. 131-49.
Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility Testing; Twentieth Informational Supplement, Document M100-S20. Wayne, PA: Clinical and Laboratory Standards Institute; 2013. p. 22-50.
Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al.
Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: An international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 2012;18:268-81.
Woodford N, Ward ME, Kaufmann ME, Turton J, Fagan EJ, James D, et al.
Community and hospital spread of Escherichia coli
producing CTX-M extended-spectrum beta-lactamases in the UK. J Antimicrob Chemother 2004;54:735-43.
Pepperell C, Kus JV, Gardam MA, Humar A, Burrows LL. Low-virulence Citrobacter
species encode resistance to multiple antimicrobials. Antimicrob Agents Chemother 2002;46:3555-60.
Rizvi M, Fatima N, Shukla I, Malik A. Epidemiology of extended spectrum beta-lactamases in Serratia
species in North India. Indian J Pathol Microbiol 2010;53:193-4.
Mohanty S, Kapil A, Dhawan B, Das BK. Bacteriological and antimicrobial susceptibility profile of soft tissue infections from Northern India. Indian J Med Sci 2004;58:10-5.
Metri BC, Jyothi P, Peerapur BV. Anti-microbial resistance profile of Citrobacter
species in a tertiary care hospital of Southern India. Indian J Med Sci 2011;65:429-35.
Brenner DJ, Grimont PA, Steigerwalt AG, Fanning GR, Ageron E, Riddle CF. Classification of citrobacteria by DNA hybridization: Designation of Citrobacter farmeri
sp. nov. Citrobacter youngae
sp. nov. Citrobacter braakii
sp. nov. Citrobacter werkmanii
sp. nov. Citrobacter sedlakii
sp. nov. and three unnamed Citrobacter
genomospecies. Int J Syst Bacteriol 1993;43:645-58.
Lavigne JP, Defez C, Bouziges N, Mahamat A, Sotto A. Clinical and molecular epidemiology of multidrug-resistant Citrobacter
spp. infections in a French university hospital. Eur J Clin Microbiol Infect Dis 2007;26:439-41.
Shahid M. Citrobacter
spp. simultaneously harboring blaCTX-M, blaTEM, blaSHV, blaampC, and insertion sequences IS26 and orf513: An evolutionary phenomenon of recent concern for antibiotic resistance. J Clin Microbiol 2010;48:1833-8.
Uma A, Mehta A, Ayagari A, Kapil A, Shahani A, Rodrigues C, et al
. Prevalence of beta lactamase producing strains among the clinical isolates which were obtained from hospital in-patients across India and comparison of the antibacterial susceptibility testing by using the disc diffusion method. Hosp Today 2004;9:1-12.
Patil MA, Lakshmi V. Antibiotic resistance among the Citrobacter
species from different clinical specimens. Indian J Med Microbiol 2000;18:25-9.
Department of Microbiology, King George's Medical University, Lucknow - 226 003, Uttar Pradesh
Source of Support: None, Conflict of Interest: None