Citrobacter braakii urinary tract infection in chronic kidney disease

Urinary tract infections (UTIs) increase with age in males and females, with incidence 50 times higher for females than for males.^1,2 Often in seniors, the symptoms are severe leading to untreatable antimicrobial resistance, organ complications and cognitive changes. UTIs have been identified as the leading healthcare-associated infections in Australia according to cross-sectional and healthcare-associated studies conducted in Australian hospitals and community healthcare centres.^3,4 Several organisms, including Pseudomonas aeruginosa, Proteus vulgaris, Proteus mirabilis, Enterobacter cloaca, Enterobacter aerogenes, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae, are responsible for causing UTIs.^5,6

Escherichia coli is the common UTI-causing microbe that often becomes untreatable due to antibiotic resistance. However, other emerging pathogens such as Citrobacter are gaining importance in hospital-associated UTIs and in people with a compromised immune system. The genus Citrobacter belongs to the Enterobacteriaceae family and is Gram-negative with peritrichous flagella.^7,8 Members of the Citrobacter genus are ubiquitous in nature; however, they have been shown to be emerging pathogens causing several infections including gastroenteritis, neonatal meningitis, septicaemia, brain abscess and UTIs.^9–11

The genus Citrobacter is a facultative anaerobic, oxidase negative, motile bacillus that grows on Simmons citrate medium using citrate as the sole carbon source.⁷ Citrobacter fails to produce acetylmethylcarbinol (VogesProskauer) and lacks lysine decarboxylase, two factors that distinguish it from other members of Enterobacteriaceae.⁸ Members of the Citrobacter genus are widely distributed in the environment, water, soil, food and intestinal tract of humans and animals.⁹ The genus is reported to be the third most common cause of UTIs, accounting for 9.4% of UTIs in hospitalised patients.^12,13 Citrobacter has been isolated in hospital settings and community-acquired infections worldwide and the emergence of multi-drug resistant (MDR) strains poses a serious challenge for clinicians.¹⁴ Overall, Citrobacter spp. have been shown to cause 5–12% of UTIs^12,15,16 and their prevalence has increased since 1961.¹² Although there were controversies around their classification, DNA–DNA hybridisation showed the DNA relatedness of strains identified⁹ genetically diverse distinguishable species within the genus Citrobacter. Genomospecies 1–3 corresponded to C. freundii, C. koseri and C. amalonaticus; genomospecies 4, which corresponded to C. amalonaticus biogroup 1, was named Citrobacter farmeri sp. nov. Genomospecies 5–8 were named C. youngae sp. nov., C. braakii sp. nov., C. werkmanii sp. nov. and C. sedlakii sp. nov. Genomospecies 9–11 were C. rodentium, C. gilleni and C. murlinae.¹⁷ C. freundii followed by C. braakii and C. koseri were the most common Citrobacter pathogens that were identified as potential disease-causing species of the genus.⁷ The predominant sites of predilection were reported to be urinary tract and the respiratory tract and often young children (<12 months old) and the elderly were found to be at risk of acquiring Citrobacter infections.⁷

Midstream urine from an 83-old female patient was presented to the Department of Molecular Microbiology, Saturn Pathology (a private pathology laboratory in Perth, WA, Australia) for microscopy, culture and antibiotic susceptibility (MCS) testing. The patient’s sample was presented with a clinical history of chronic kidney disease and full blood picture, renal function tests and liver function tests were also requested along with MCS testing. Urine dipstick analysis (Abbott, UroColor 10, Australia) and microscopy (Olympus, USA) of the urine sample were carried out and a semiquantitative urine culture using a 1-µL calibrated loop (MWE, Medical Wire, Australia) was performed by inoculating cystine–lactose–electrolyte deficient (CLED) agar (PathWest Media, Perth, WA, Australia) for bacterial differential culture and enumeration as a default routine urinalysis. A single colony from the CLED agar plate was sub-cultured onto sheep blood agar for purification, identification and antibiotic susceptibility tests (ASTs). An Analytical Profile Index 20E (API20E) strip (bioMérieux Australia, Table 1) was used to identify the isolate by suspending pure culture in 0.85% saline (PathWest Media) equilibrated to 0.5% McFarland standard (PathWest Media). Oxidase test (Oxoid, ThermoFisher, Australia) was carried out using oxidase strips by smearing a portion of the culture on to the strip as per manufacturer’s protocol, and a formation of blue colour within few seconds was analysed and recorded as observed. Further the isolate was identified using MALDI-TOF MS (Bruker, tof-user@FLEX-PC, type: standard, instrument ID: 8269944.03039, server version: 4.1.100 (PYTH) 174 2019-06-158_01-16-09). Antimicrobial susceptibility testing was carried out following Kirby–Bauer disc diffusion method.¹⁸ The antimicrobials (Oxoid, ThermoFisher) tested are listed in Table 2 with results. Results from urine dipstick showed 1+ red blood cells (RBCs), 3+ leucocytes, negative bilirubin, urobilinogen, ketones, glucose and nitrites with a trace of protein. Direct wet microscopical analysis revealed white blood cells to be >100 µL^–1, RBCs 20–50 µL^–1, epithelial cells 5–10 µL^–1 and no casts or crystals, but numerous bacteria. On CLED agar plate after 24 h of incubation, yellow-coloured medium-sized irregular or rough-edged colonies accounting for >10⁸ colony forming units mL^–1 were isolated. The morphological characteristics were similar to E. coli in appearance and colour; however, the colonies were not smooth as E. coli. API20E results were read following the manufacturer’s instructions and the profile number was obtained by entering the results on the APIWEB website (see https://www.biomerieux.com/). The isolate obtained an analytical profile index profile with 99.9% accuracy for Citrobacter braakii: 3544553. MALDI-TOF identified the isolate as Citrobacter braakii with an identification score value of 2.0, which is considered an accurate level of species identification.¹⁹ Table 1 shows the reaction results from the API20E strip for the positive control organism procured through Royal College of Pathologists of Australasia Quality Assurance Programs (RCPA-QAP).

Biochemical test (Abbot, Architect, Australia) results showed raised albumin : creatinine ratio (36.6 mg mmol^–1), uric acid (0.50 mmol L^–1), total cholesterol (5.7 mmol L^–1), triglycerides (2.89 mmol L^–1), LDLC (3.5 mmol L^–1), HDL-C (1.0 mmol L^–1), globulins (45 g L^–1), parathyroid hormone (10.1 pmol L^–1), protein creatinine ratio (93 mg mmol^–1), urea (14.1 mmol L^–1), creatinine (209 μmol L^–1) and decreased estimated glomerular filtration rate (18 mL/min/1.73 m²), magnesium (0.68 mmol L^–1) and bicarbonate (21 mmol L^–1). Blood parameters showed increased lymphocyte counts 4.89 (54.9%), decreased RBC count 3.54 (10¹² L^–1) and decreased hemoglobin (95 g L^–1).

In our report, the sample was from an 83-year-old female with chronic kidney disease, which is in agreement with other findings in terms of age being a risk factor for acquiring Citrobacter infection and the predilection site, the urinary tract.^7,12,15,16 Often Citrobacter spp. identification can be challenging due to the lack of accurate identification methods and can go misidentified. For example, it is reported that C. braakii is misidentified due to the usage of conventional methods²⁰; however, in our report we identified the isolate using API and MALDI-TOF MS, whereas 16S rRNA has also been used for identification.²⁰

Although, Citrobacter spp. are regarded as opportunistic pathogens, variable prevalence of extended spectrum β-lactamases (ESBLs) producing Citrobacter strains have been reported from South Korea, Japan and USA; and 3.5 and 60.0% of C. koseri isolates from USA and Japan respectively^21,22 and CTX-M types, SHV and TEM Citrobacter types have been reported worldwide.²³ The ampicillin-resistance pattern in our study is in agreement with previous reports,^7,24 where Arens et al.⁷ reported a 4% ampicillin and amoxicillin–clavulanate-susceptible isolates and a 8% cefazolin-susceptible isolates out of 25 C. braakii isolates tested; Liu et al.²⁴ reported 71% of C. braakii isolates exhibiting resistance to ampicillin followed by ceftazidime resistance (43%) and ciprofloxacin resistance (14%). However, the isolate in our report was susceptible to ceftazidime and ciprofloxacin. Antibiotic-susceptibility results of C. braakii are sparce and there are no resistant strains to carbapenem (imipenem and meropenem), fosfomycin or sulfamethoxazole–trimethoprim reported including our report so far.²⁰ However, it should be noted that plasmid-mediated quinolone-resistance genes including qnr and aac(6′)-Ibcr have been reported in C. freundii isolates²⁵ and we speculate that C. braakii may evolve to possess multi-drug resistance mediated by either mutation or by plasmids given it belongs to the C. freundii complex.

It should be acknowledged that the risk factors, epidemiology and clinical features of C. braakii infections are still unclear.²⁰

In conclusion, we recommend that the isolates from the same site should be tested and confirmed for ASTs after 3–4 days of commencement of treatment, as Citrobacter spp. tend to develop resistance to next-generation cephalosporins due AmpC β-lactamases.¹⁸ We also recommend that a full biochemical profile and MALDI-TOF identification be performed to differentiate Citrobacter spp. from E. coli and to prevent inaccurate antimicrobial-susceptibility interpretation. Reporting of not-so-common pathogens in a timely manner will help to provide insights into emerging pathogens in hospital settings and healthcare-associated infections. This in turn will keep the clinicians and specialists informed about the antibiotic usage and emergence of MDR strains that have a massive effect on the human health sector.

Ethics

Saturn Pathology performs home collections, a phlebotomist visits patients on request and prebookings with the pathology lab and the patients are requested to give their consent to collect samples and for pathology services. The patient’s details such as their names and residence are kept anonymous.