Prevalence and antimicrobial susceptibility pattern of Citrobacter species in various clinical samples in a tertiary care hospital

Background: Infections with Citrobacter species have been increasing across worldwide. Earlier, considered to be organism of low virulence but now known to cause variety of infections with high morbidity and mortaliy. Objective: The present study aims to determine the prevalence and antibiotic susceptibility pattern of Citrobacter species from various clinical samples in a tertiary care hospital. Material and methods: This one year prospective observational study, conducted in the Department of Microbiology at Krishna Institute of Medical Sciences, Secunderabad from January 2018 to December 2018. The samples received to the laboratory from both outpatient and inpatient from various departments. Identification and antimicrobial susceptibility of the isolates were done by using Vitek 2 compact automated system (bioMerieux). Results: In 3,337 culture positive sample, 83 (2.4%) isolates were found to be culture positive for Citrobacter species where C. koseri (n=57) is predominant followed by C. freundii (n=24) and C. amalonaticus (n=2). Majority of Citrobacter species were isolated from urine samples 56% followed by pus 21%, sputum 8.43%, blood 7.2% and in bile fluid 3.6%. The highest incidence was found in elderly patients of age group 51-80 year age group, more common in males (70%) than females (30%). Conclusion: The present study highlight that Citrobacter species are an emerging pathogen with prevalence of 2.4%. It is the most common uropathogen followed by pus and sputum, and bacteraemia also. C. freundii was comparatively less susceptible as compared to C. koseri. Antibiotic policy should be implemented strictly and followed strongly so that emergence of such low virulent organism with emerging resistance can be controlled.


Introduction
Infections with Citrobacter species have been increasing across worldwide [1]. Citrobacter species belong to a group of facultative anerobic, gram-negative bacilli belongs to the family Enterobacteriaceae, tribe Citrobactereae [2]. It is found singly or in pairs, and are motile by peritrichous flagella which typically utilize citrate as sole source of carbon [3]. In the environment, they are frequently found in water, soil, food, and as an occasional colonizers of the intestines of human and animals. These organisms are isolated from variety of clinical specimens like urine, pus, blood, and cerebrospinal fluid [4].
Earlier, they are recognised as environmental contaminants or colonizers with low virulence, they are now known to cause variety of infections such as the urinary tract, wounds, soft tissue, respiratory tract, liver, bloodstream, biliary tract, peritoneum, meningitis and brain abscess [6].
Although Citrobacter infections occur in adults with immunocompromised state but it is now an emerging cause of UTIs ,neonatal septicaemia, neonatal meningitis, brain abscess with high mortality as high approximately one-third of infants with abscesses die, and one-half sustain CNS damage [7,8].
Citrobacter infections were found to represent 0.8% of Gram-negative infections in a large surveillance study conducted in American medical centres [9], while, in hospital settings, accounted for 3-6% of all isolates of Enterobacteriaceae [9]. There are reports of Citrobacter species being a major nosocomial pathogen with multi drug resistant antimicrobial susceptibility [10]. The present study was taken up to determine the prevalence and antibiotic susceptibility pattern of Citrobacter species from various clinical samples in a tertiary care hospital.

Material and methods
This was a prospective observational study, conducted in the Department of Microbiology at Krishna Institute of Medical Sciences, Secunderabad from January 2018 to December 2018. A total of 17,544 samples received to the laboratory from both outpatient and inpatient from various departments. Out of total samples, 3,337 were culture positive. The samples included urine (clean catch & catheter catch), pus, and sputum, endotracheal tube secretions, sterile body fluids and blood. All the samples were received in the microbiology laboratory under sterile conditions and were processed as per standard microbiological guidelines.
Specimens were processed on blood agar (BA), Mac Conkey agar (MA) plate and urine samples were processed on cysteine lactose electrolyte deficient (CLED) agar. The inoculated culture plates were incubated at 37 o C for 16-18 hours under aerobic conditions. In case of blood sample, paired samples were collected and they were inoculated in BacT Alert bottles which were incubated in BacT Alert 3D (bioMerieux).
After culture growth, bacterial identification and susceptibility was done by VITEK 2 compact (bioMerieux) automated system. Identification of the isolates were done by using Vitek GN-ID card and antimicrobial susceptibility testing was done by using GN AST 280 card.

Results
In 3,337 culture positive sample, 83 (2.4%) isolates were found to be culture positive for Citrobacter Vol. 7 | Issue 4 | October -December 2019 species where C. koseri (n=57) is predominant followed by C. freundii (n=24) and C. amalonaticus (n=2). Majority of Citrobacter species were isolated from urine samples 56% (n=47) in which clean catch is (n=37) 44% is high as compared to catheter catch (n=10) is 12% followed by pus (n=18) 21% , sputum (n=7) 8.43%, blood (n=6) 7.2% and in bile fluid (n=3) is 3.6% as shown in Table 1. The highest incidence was found in elderly patients of age group 51-80 year age group, more common in males (70%) than females (30%) as shown in Table  2. The susceptibility pattern of various antimicrobials used in the Vitek 2 compact for each Citrobacter species isolated is given in Table 3 and Figure 1.
Citrobacter spp inherit intrinsic resistance to certain antimicrobials. C. koseri, C. fruendii, and C. amalonaticus are intrinsically resistant to amipicillin whereas C. fruendii is intrinsically resistant to amoxicillin-clavulanate, cephalosporin I and II . Therefore, these antimicrobials were excluded while reporting for susceptibility [11].

Discussion
The present study indicates the importance of Citrobacter species in various clinical samples as they are becoming an emerging pathogen with prevalence of 2.4% in 1 year. In the present study it has emerged as a potential uropathogen 56% followed by pus 21.6%, sputum and blood with 8.4% and 7.2% respectively.
The majority of age group affected in the present study is elderly age group of 51-80 years with male predominance which is similar to Metri et al. [13] and Samsonis et al. [6]. The reason of this could be because of the fact that these group of population have reduced immunity, having co-morbid diseases and more prone to hospitalisation.
In the present study the most common isolate identified is C. koseri (68.60%) followed by C. freundii (29%) and C. amalonaticus (2.40%) therefore Antimicrobial susceptibility (AST) pattern was compared between C. koseri and C. freundii as C. amalonaticus isolated is too low to be commented upon.
Aminoglycosides in case of C. koseri showed amikacin and gentamycin of good susceptibility of 96% and 98% each as compared to 79% and 70.8% in case of C. freundii.
In cephalosporins 3 rd generation ceftriaxone showed 75.4% and 62.5% which is pretty low as compared to 4 th generation cefepime which showed 92% and 87.5% in C. koseri and C. freundii respectively. In fluoroquinolones, ciprofloxacin showed 87% and 75 % susceptibity in C. koseri and C. freundii respectively.
Various studies observed the increased resistance in Citrobacter species in different parts of India but in the present study it shows considerable good susceptibility. This can be due to wide variation in specimen considered, hospital environment and hospital infection control policy.

Conclusion
The present study highlights that Citrobacter species are an emerging pathogen with prevalence of 2.4%. It is the most common uropathogen followed by pus and sputum, however it is now shown to cause bacteraemia also. In this study, C. freundii was comparatively less susceptible as compared to C. koseri therefore more studies are required to keep the check on changing susceptibility pattern. The only limitation of the present study is isolation of C. amalonaticus is too less to comment on its susceptibility pattern. Hospital antibiotic policy should be implemented strictly so that emergence of such low virulent organism with emerging resistance can be controlled. Most importantly, strict adherence to infection prevention measures, implementation of antimicrobial stewardship programme and maintaining proper hand hygiene practices in tertiary care hospitals should be followed to prevent the transmission of this emerging pathogen.

Conflicts of interest
Authors declare no conflicts of interest.