The Emergence of the Genus Comamonas as Important Opportunistic Pathogens

Comamonas spp. are non-fermenting Gram-negative bacilli. They were first discovered in 1894, and since then, twenty-four species have been characterized. The natural habitat of these bacteria is soil, wastewater/sludge, fresh water such as ponds and rivers, and the animal intestinal microbiome. They were also isolated from industrial settings, such as activated sludge and polluted soil, and from the hospital environment and clinical samples, such as urine, pus, blood, feces, and kidney. Comamonas spp. are associated with environmental bioremediation and are considered an important environmental bacterium rather than a human pathogen. However, in the 1980s, they became a concern when several human infections associated with these species were reported. Here, the Comamonas genus was examined in terms of its members, identification techniques, and pathogenicity. Seventy-seven infection cases associated with these microorganisms that have been discussed in the literature were identified and investigated in this project. All relevant information regarding year of infection, country of origin, patient information such as age, sex, underlying medical conditions if any, type of infection caused by the Comamonas species, antibiotic susceptibility testing, treatment, and outcomes for the patient were extracted from case reports. The findings suggest that even though Comamonas spp. are thought of as being of low virulence, they have caused harmful health conditions in many healthy individuals and even death in patients with underlying conditions. Antimicrobial treatment of infections associated with these species, in general, was not very difficult; however, it can become an issue in the future because some strains are already resistant to different classes of antibiotics. Therefore, these pathogens should be considered of such importance that they should be included in the hospital screening programs.


Introduction
The growing range of severe infections caused by little-known non-fermenting Gramnegative rods is developing into a major cause of concern. These pathogens are opportunistic, infecting patients undertaking medical treatments in hospital and immunocompromised individuals outside of clinical locations. Bacterial species, including Ralstonia spp., Ochrobactrum spp., Pseudomonas aeruginosa, Sphingomonas paucimobilis, and Brevundimonas spp., all belong to this group [1][2][3][4][5][6]. Other emerging Gram-negative, non-fermenting rod bacteria that can cause potentially severe infections are members of the β-proteobacterial genus Comamonas [7].
Comamonas spp. have been isolated from a broad variety of environments, including water, aircraft water, soil, plants, and animals [8][9][10][11][12]. Several Comamonas spp. have been investigated for their potential to degrade xenobiotic pollutants and for heavy metal detoxification under a variety of environmental conditions [13][14][15][16][17][18][19]. Comamonas spp. are thought to be of low virulence. They have, however, caused infections, including serious infection such as septicemia or endocarditis, in immunocompetent hosts [20][21][22].  . Phylogenetic tree of the genus Comamonas (accession numbers are given alongside species name) with the closely related genus Delftia. The tree was built with 16S rDNA genes (partial sequences of~1400 bp) using neighbor-joining with the Tajma-Nei method utilizing the MEGA 11 software package. Bootstrap values are represented by numbers at nodes. These are based on 1000 resamplings. Bar, 0.0050 substitutions per site [26,27]. It should be remembered that these analyses are based upon 16S rDNA and, as such, are suggestive only.

Identification of Comamonas spp.
The Comamonas species are Gram-negative and comprised of straight or slightly curved rods or spirilla. They are usually 0.5 to 2 by 1 to 6 µm. They are generally motile by means of polar or bipolar tufts of 1-5 flagella (excepting C. koreensis). They are aerobic and chemoorganotrophic (De Vos et al., 2015) [50]. Some of the species are non-pigmented, some appear to be cream or yellow-white in color, and some can produce a brown halo around them (Willems and De Vos, 2006) [51], but they do not produce fluorescent pigments. Colonies appear pink-pigmented with a slimy and convex surface on blood agar. No hemolysis was observed on blood and chocolate agar. They are aerobic, oxidase and catalase-positive, non-spore formers, glucose non-fermenters, and chemoorganotrophic. Good growth was observed on media that contained peptone, organic acids, and amino acids (Public Health England, 2015) [52].

Comamonas spp. Virulence
Comamonas spp. are believed to be of low virulence. A study of the pangenome of 34 Comamonas genomes, however, showed that they have a diverse array of virulence factors, including polysaccharide biosynthesis for adherence and anti-phagocytosis, a motility system and metabolic enzymes for adaptation in vivo. All sequenced, clinicallyisolated Comamonas strains and a number of environmental Comamonas spp. contain hemolysin genes. These analyses indicated that virulence might be species-specific as certain virulence factors are conserved in pathogenic-like strains [53].

Comamonas spp. Outbreaks
The overall knowledge gained from research into the scientific and medical literature can be seen in Tables 2-4. These tables show the year when the infection happened (if not available, the year of publication was used), country where the infection happened, patient information (age, sex, any reported underlying medical conditions), type of infection caused by the Comamonas infection, antimicrobial testing (susceptibility and resistance), treatment (focusing on the antibiotic therapies used) and patient outcome.
Most patients had one underlying condition, seven had patients with two underlying conditions, and eight had patients with multiple underlying conditions (for example, obesity and diabetes). The most abundant of these underlying conditions were diabetes (in 8 patients-10.3%), various types of cancer (in 5 patients-6.5%) and alcoholism (in 4 patients-5.2%). Other major underlying conditions included obesity (in 3 patients-3.9%), hypertension (in 4 patients-10.9%), and renal failure (in 3 patients-3.9%). A full list of underlying conditions can be seen in Tables 2-4. A total of 70 patients (92.1%) were treated successfully and recovered fully, and 6 patients (7.8%) died. All patients who died due to Comamonas spp. infection suffered from one or more underlying conditions. These cases are discussed in more detail below. Surprisingly, to date, no pseudo-outbreaks have been found associated with Comamonas spp.
Most of the reported infections caused by Comamonas spp. appear to be communityacquired [22].

Death Associated with Comamonas spp. Infection
Six instances of death associated with Comamonas spp. infection have been reported. All six cases were linked to C. testosterone ( Table 2). The first two instances were reported by Barbaro et al. [54]. In one of these instances, a mother who was an intravenous drug abuser gave birth to a premature baby, and this newborn baby died of sepsis caused by C. testosteroni infection 24 h after he was born. The second instance was very similar as it was also associated with sepsis due to C. testosteroni infection in a premature baby who was stillborn by an intravenous drug abuser mother. The third instance of death was reported in 2008 by Jin et al. [55]. In this case, a 54-year-old homeless man alcoholic was hit by a car, he received multiple fractures of the facial bones and was hospitalized. He was diagnosed with multiple cerebral and cerebellar infarcts, which resulted in changed mental status. He died 15 days after the injury. An autopsy revealed diffuse purulent meningitis due to C. testosteroni infection. In the fourth instance reported by Swain and Rout, a 50-year-old woman who suffered from diabetes and had a chronic renal disease was hospitalized for bacteremia and septic shock [56]. She was treated with piperacillin-tazobactam antibiotics until C. testosteroni was identified. The microorganism was found to be resistant to piperacillin-tazobactam, so treatment was then changed to cefoperazone-sulbactam. However, despite this, the woman died due to septic shock. The fifth instance of death associated with Comamonas spp. was reported in 2017 by Yasayancan and Koseoglu [57]. A 68-year-old man with lung cancer and adrenal metastasis was diagnosed with polymicrobial bacteremia due to C. testosteroni, Staphylococcus haemolyticus, and Acinetobacter baumannii infection. The patient died on the 16th day, despite suitable treatments against these pathogens. The last reported instance of death due to C. testosteroni infection was reported in 2018 by Cetin et al. A 10-year-old boy with serious underlying conditions (cerebral palsy, scoliosis, and long-term support with home mechanical ventilation) was diagnosed with pneumonia due to C testosteroni infection [58]. The patient was treated with appropriate antimicrobial therapy, and after 21 days of treatment infection was cured but due to the patient's poor health conditions, he died on day 50 of hospitalization. No deaths have been associated with C. kerstersii or any other Comamonas spp (Tables 3 and 4).

Treatment of Comamonas spp. Infections
Antibiotic treatment of Comamonas spp. infections can be difficult. Comamonas spp. can be resistance to various antibiotic families including β-lactams (penicillins, cephalosporins and the development of resistance to carbapenems). To date, no controlled trials of antimicrobial therapies for Comamonas spp. infections in humans have taken place; consequently, antibiotic treatment ought to be based upon results of in vitro susceptibility testing on isolates. A variety of different antibiotics have been employed to treat Comamonas spp. infections found in the literature and, in most cases, they are susceptible to aminoglycosides, fluoroquinolones, carbapenems, piperacillin-tazobactam, trimethoprim-sulfamethoxazole, and cephalosporins (Tables 2-4).
Resistance to β-lactams class antimicrobials can be due to the possession of several genes by Comamonas spp. C. testosteroni S44 possesses a three-gene operon that codes for a Class A β-lactamases (resistance to benzylpenicillin, ampicillin, cefalexin, cefazolin, cefuroxime, ceftriaxone, and cefepime). These genes are CzoA (Class A β-lactamase encoding gene)-inhibits β-lactams antibiotics, CzoR (LysR type transcriptional regulator)positively affects the expression of CzoA, and the IscR gene-enhances the regulatory effect of CzoR when bounded to its promoter region [106]. Several resistance genes were found in C. kerstersii 8943, including tetA, strB, sul1, bla OXA-1 , strA, sul2, catB3 and floR. The bla IMP-8 gene (giving resistance to β-lactam antibiotics) has been found in a Comamonas thiooxydans isolate, which caused a urinary tract infection. This isolate also had a novel class D betalactamase gene bla OXA and a aac(6 )-Ib-c gene (resistance to aminoglycoside antibiotics). A variety of efflux pumps were also identified in the genomes of this bacterial isolate. [105]. A study in 2022 found another Comamonas thiooxydans isolate with a plasmid-based bla IMP-1 gene [107]. In a study by Hem et al., 2022, 32 Comamonas. denitrificans and 5 C. testosteroni from wastewater, 1 C. denitrificans from a wetland, and 1 C. aquatica from a lake with public access were sequenced. All were found to be resistant to carbapenem antibiotics. However, only 13 C. denitrificans isolates were found to have an identifiable carbapenemase bla  . No identifiable carbapenemase genes were found in the other isolates. Other C. denitrificans isolates carried extended-spectrum b-lactamase (ESBL) bla OXA genes. This was the first report of resistance to carbapenem antibiotics in both C. denitrificans and C. aquatica; however, carbapenem-resistance was previously reported in a C. testosteroni infection in Turkey in 2015 [77,108].

Conclusions
Comamonas spp. are not currently considered important pathogens and are thought of as being of low virulence and of being a lesser danger in comparison to other nonfermenting Gram-negative bacteria such as Pseudomonas aeruginosa. Nevertheless, in this review, fifty-five separate outbreaks of Comamonas spp. infections have been identified from the scientific literature not taking into account unreported/undocumented cases. It must be recommended that the scientific community acknowledge the ability of this organism to elude antimicrobials and thus the potential for antimicrobial resistance transference between organisms, particularly in an era of growing antimicrobial susceptibility concerns.

Conflicts of Interest:
The authors declare no conflict of interest.