Epidemiology of Raoultella species in the context of human infections: A 10-year retrospective study in a tertiary-care hospital in Hungary

Introduction: Raoultella species are Gram-negative non-motile, aerobic, oxidase-negative and catalase-positive rods that are members of the Enterobacterales order that are infrequently described as significant pathogens in human disease. Objectives of the study: To report the incidence and the susceptibility levels of Raoultella species from clinical samples at a tertiary-care center in Hungary, during a 10-year study period (2008–2017). Method: Data collection was performed electronically by searching the records of the laboratory information system (LIS), regarding the samples with significant colony counts for Raoultella spp. Results: N=40 individual Raoultella isolates were identified (4.0±2.8/year, range: 0–9 isolates; highest in 2015, lowest in 2010 and 2011). Raoultella species were isolated from the following sample types: urine (catheterized and midstream): n=18, blood cultures: n=12, stool: n=6 and abscesses: n=4. 36 out of 40 isolates were R. ornithinolytica , while the remaining n=4 were R. planticola . All of the tested strains were susceptible to the group of β -lactam antibiotics. Conclusion: Raoultella species should be considered as emerging pathogens in the context of human infections, especially in older individuals in people with severe immunosuppression. The results suggest that in our local settings, there was-on average-one isolation of Raoultella every three months from various clinical samples and β -lactams may be considered safe choices for empiric therapy.


Introduction
Raoultella species are Gram-negative non-motile, aerobic, oxidase-negative and catalase-positive rods that are members of the Enterobacterales order (gut bacteria) [1,2]. The genus Raoultella was previously designated to the genus Klebsiella (as cluster II; e.g., K. ornithinolytica), but after extensive advancement in phylogenetic methods (16S rRNA and rDNA sequencing, bacterial RNA polymerase β subunit [rpoB] sequence-analysis), the genus Klebsiella was revised and the independent genus Raoultella was created in 2001 (named after Didier Raoult, a French bacteriologist) [3,4]. R. ornithinolytica is the most abundantly found both in clinical samples and in nature (in aquatic environments, soil and in different animals, such as fish, insects and ticks); however R. terrigena, R. planticola and R. trevisanii are also important members of the genus [2][3][4].
The existing literature on Raoultella infections in the context of human infections is scarce, as the incidence of these pathogens in clinically-relevant syndromes in low [5]. Most existing reports on these bacteria are associated with infections of the gastro-intestinal, hepatobiliary, urological and respiratory systems, while bloodstreaminfections, osteo-articular, soft tissue and ear-nose-throat (ENT) infections have also been published [6][7][8][9][10][11]. It should be noted, that the difficulty in the adequate identification (especially in low-resource settings) may be partly blamed for the infrequent characterization of these bacteria as significant pathogens [12]. Some reports even suggest that around 20% of Raoultella species are misidentified as Klebsiella (predominantly K. oxytoca) [13]. Nevertheless, with the advent of novel diagnostic modalities in routine use (such as whole-genome sequencing [WGS] and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry [MALDI-TOF MS]) and reports on the emergence of multidrug-resistant strains of Raoultella, the clinical role of this genus should not be underestimated [14]. The aim of this study was to report the incidence and the susceptibility levels of Raoultella species from clinical samples at a tertiary-care center in Hungary, during a 10year study period (2008-2017).

Study design and data collection
The present retrospective study was carried out using microbiological data collected corresponding to the time period between 2008.01.01.-2017.12.31., at the Institute of Clinical Microbiology, University of Szeged. The Institute serves as the dedicated diagnostic institute of the Albert Szent-Györgyi Clinical Center, which is a 1,820-bed primary-and tertiary-care teaching hospital in the Southern Great Plain of Hungary (with an estimated population of over 400,000 people, based on data from 2017) [15]. Data collection was performed electronically by searching the records of the laboratory information system (LIS), regarding the samples with significant colony counts for Raoultella spp. (>10 5 CFU/mL for urine samples, while >10 3 in case of other types of clinically-relevant samples; however, this was subject to interpretation by the senior clinical microbiologists, based on the information provided on the clinical request forms for the microbiological analysis and international guidelines) [16]. Only the first isolate per patient was included in the study; however, isolates with different antibiotic-susceptibility patterns from the same patient were considered as different individual isolates. To evaluate the demographic characteristics of these infections, patient data was also collected, which was limited to sex, age at sample submission, and inpatient/outpatient status [16]. The study was deemed exempt from ethics review by the Institutional Review Board and informed consent was not required as data anonymity was maintained.

Sample processing and identification
The processing of relevant samples arriving to the Institute of Clinical Microbiology was carried out according to guidelines in routine clinical bacteriology. Between 2008-2012, the BD Bactec (Beckton Dickinson, Franklin Lakes, NJ, USA) detection system was employed for the incubation of blood culture bottles, whilst from 2013 onwards, the BacT/ALERT 3D (bioMérieux, Marcy-l'Étoile, France) detection system was used. Blood culture bottles were incubated for 5 days (21 days, if endocarditis was suspected) in the abovementioned detection systems [17]. The processing of urine samples was as follows: 10 µL of each un-centrifuged urine sample was cultured on UriSelect chromogenic agar plates (Bio-Rad, Berkeley, CA, USA) with a calibrated loop, according to the manufacturer's instructions and incubated at 37°C for 24-48 h, aerobically [15]. The workup of faecal samples was performed on the appropriate non-selective and selective media. If the relevant pathogens presented in significant colony count, the plates were passed on for further processing.
Between 2008-2012, presumptive phenotypic (biochemical reaction-based) methods and VITEK 2 ID (bioMérieux, Marcy-l'Étoile, France) were used for bacterial identification, while after 2013, this was complemented by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; Bruker Daltonik Gmbh. Gr., Bremen, Germany). Bacterial cells were transferred to a stainless-steel target. An on-target extraction was performed by adding 1 ml of 70% formic acid prior to the matrix. After drying at ambient temperature, the cells were covered with 1 ml matrix (α-cyano-4hydroxy cinnamic acid in 50% acetonitrile/2.5% trifluoro-acetic acid). Mass spectrometry was performed by the Microflex MALDI Biotyper (Bruker Daltonics, Bremen, Germany) in positive linear mode across the m/z range of 2 to 20 kDa; for each spectrum, 240 laser shots at 60 Hz in groups of 40 shots per sampling area were collected [18]. The MALDI Biotyper RTC 3.1 software (Bruker Daltonics) and the MALDI Biotyper Library 3.1 were used for spectrum analysis.

Statistical analysis
Descriptive statistical analysis (including means or medians with ranges and percentages to characterize data) was performed using Microsoft Excel 2013 (Redmond, WA, Microsoft Corp.).

Results
During the 10-year study period, a total of 40 individual Raoultella isolates were identified All of the tested strains were susceptible to the group of β-lactam antibiotics (40 out of 40; from ampicillin to meropenem, respectively), while 8/40 of isolates were resistant to norfloxacin, 6/40 to ciprofloxacin and levofloxacin, 5/40 to sulfamethoxazole/trimethoprim and 2/60 to the tested aminoglycosides, respectively. No ESBL-or AmpC-producing strain was detected during the study period.

Discussion
The present study reports on the epidemiological features of Raoultella infections at a tertiary-care hospital in Hungary over a period of a decade (2008-2017). Due to the developments in diagnostic technologies in routine clinical microbiology (e.g., MALDI-TOF MS), the prevalence of bacterial species that were previously considered as rare will most probably increase, which is reflected in the increase in the interest towards these bacteria in the literature [1][2][3][4][5][6][7][8][9][10][11]19]. The clinical role and prognosis of Raoultella infections highly depend on the health status of the patient: the risk of sequelae is higher in older, co-morbid patient with invasive infections, therefore the awareness of the clinicians regarding the epidemiology and susceptibility-levels of these bacteria is of utmost importance [20][21][22]. In case of those patients, the initiation of prompt antibiotic therapy is crucial for recovery, while in immunocompetent patients, symptoms may resolve with supportive therapy as well [1][2][3][4][5][6][7][8]. The virulence factors of Raoultella are thought to be similar to the ones of Klebsiella species due to their phylogenic similarities, however, no such correlation was found in vivo [12,23].
The limitations of this study should be reported: the study was retrospective, thus the symptoms of individual patients affected by these infections could not be recorded in real-time. Additionally, the molecular characterization of resistance determinants in the mentioned isolates was not performed; therefore non-susceptibility was characterized by phenotypic methods only [15].

Conclusions
Although infrequently isolated (or identified), Raoultella species should be considered as emerging pathogens in the context of human infections, especially in older individuals in people with severe immunosuppression. The results suggest that in our local settings, there was-on average-one isolation of Raoultella every three months from various clinical samples and β-lactams may be considered safe choices for empiric therapy of these infections, taking into account that the emergence of resistant mutants is possible. The surveillance of rarely isolated species over long periods of time may help ascertain their true clinical role in human infections.

Author contributions
M.G. conceived and designed the study, performed data collection and analysis, wrote and revised the full paper.

Funding
This research received no external funding.