Indian Journal of Pathology and Microbiology

: 2014  |  Volume : 57  |  Issue : 3  |  Page : 439--441

Observations on Citrobacter species from a tertiary care health center with special reference to multi-drug resistance and presence of CTX-M gene

Sneha Mohan, Jyotsna Agarwal, Richa Srivastava, Mastan Singh 
 Department of Microbiology, King George's Medical University, Lucknow, Uttar Pradesh, India

Correspondence Address:
Jyotsna Agarwal
Department of Microbiology, King George«SQ»s Medical University, Lucknow - 226 003, Uttar Pradesh


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.

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;57:439-441

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 Dec 4 ];57:439-441
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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.[1],[2] 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. [3] 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. [4],[5] 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.


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. [6]

Antibiotic sensitivity testing

Antibiotic susceptibility of isolates was done on Muller Hinton agar using Kirby Bauer method (Clinical and Laboratory Standards Institute [CLSI] 2013). [7] 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. [8]

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). [7] 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) [7] 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. [9]


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.{Table 1}


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. [10] Very few studies in India have dealt with Citrobacter and its antibiotic sensitivity pattern. [1] 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. [1],[2] While few others have reported C. koseri to be the predominant species. [11],[12],[13] 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. [14] Maximum number of isolates were received from surgical ward (46.9%) followed by ICU (30.4%) similar to a previous study. [13] 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. [15]

Extended spectrum beta lactamase production was found in 54% (79/146) isolates, which was similar with the finding of Khanna et al. [1] (57%) and Rizvi et al. [11] (62%), but much lower than that reported by some other studies. [16],[17] Indiscriminate and overuse of extended spectrum antibiotics is shown to be the common reason for high prevalence of MDR and ESBL in hospital setup. [16],[18] 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. [16] 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. [4] Proper aseptic and barrier precaution along with appropriate antibiotic policy are needed to prevent dissemination of such resistant strains in hospital setup.


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