In the last few years, there have been works in literature describing clinical cases of infections with bacteria previously considered to be non-pathogenic. So far, these bacteria have been commonly isolated from the natural environment: water, soil, plants, and isolation from a sample of clinical material has often been considered an impurity. Therefore, the aim of the paper was to draw attention to newly emerging bacterial species, their characteristics, biochemical properties, virulence factors and predisposition to selected forms of infection in groups of patients with specific risk factors. The impact on the more frequent isolation of species of bacteria previously considered to be non-pathogenic has several aspects: reliable diagnostic methods, including correct identification of the species, enabling the differentiation of closely related bacteria, the growing group of immune-compromised patients and with infectious risk factors (such as older age, diabetes, tumours, organ transplantation), more susceptible to infection, also with less pathogenic microorganisms. Greater adaptability of bacteria to new environmental conditions, including the human body, are also worth mentioning. The pathogenic effect of these bacteria, mainly related to the production of biofilms and factors conditioning adhesion to host cells or biomaterials, facilitating colonization and subsequent infection, is also important.
The progress in medicine that has taken place in recent years has positively influenced the life expectancy of patients with certain diseases and in many situations, it contributed to an improvement in its quality. Unfortunately, it also made it possible to cause infection with bacteria that were previously considered to be low or non-pathogenic.
1. Introduction. 2. Gram-positive rods 3. Streptococci with special nutritional requirements 4. Gram-negative rods. 5. Summary
More frequent and wider application of quick methods for identifying microorganisms based on new technologies in microbiological diagnostics has led to the appearance of descriptions of bacterial infection cases in literature which have been so far disregarded due to the impossibility of their description or have been considered to be scarcely virulent or non-pathogenic. New methods include, among others, mass spectrometry or infrared spectrometry. These methods are highly sensitive and specific, as well as simple to perform. In addition, the waiting time for the identification result from the sample setting in the case of mass spectrometry is 15 minutes, or even shorter. This has a significant value in the diagnostic and therapeutic process as well as in the economic aspect. An advantage of these methods is also the possibility of differentiating microorganisms being closely related phylogenetically. Also, the development of molecular biology methods, allowing the study of the similarity between bacteria, has caused changes in the systematics and reclassifications within previously known families and genera. These bacteria are commonly found in the natural environment: in water, soil, on plants, in the air. They have also been isolated from animals.
The aim of the work was to draw attention to newly emerging bacterial species, which are increasingly often isolated in human infections. The work includes data from our own diagnostic practice and from the available literature.
The newly appearing bacterial species belong to morphologically different groups of bacteria. Among the Gram-positive rods, the genera: Arcanobacterium and Brevibacterium are increasingly often isolated. Arcanobacterium spp. is non-motile, non-sporing, pleomorphic rods. This genus was reclassified from the Corynebacterium genus in 1982 . This bacterium possesses characteristic fatty acids in the cell wall; it does not produce catalase and belongs to facultative anaerobes. In the microscopic picture it takes the form of irregular cells, arranged in groups of two or three resembling the letters T, V, L, X, Y. The genus Arcanobacterium includes the species Arcanobacterium haemolyticum . On solid substrates, this bacterium can form two types of colony morphology: smooth and rough. Most strains form smooth colonies. Strains with such colony appearance trigger beta-haemolysis, do not produce β-glucuronidase, and ferment sucrose and trehalose. Strains with rough colonies do not induce haemolysis, produce β-glucuronidase and do not ferment sucrose and trehalose. Strains with smooth colonies are more likely to cause skin infections, while the rough ones, in most cases of infections, are isolated from the material originating from the respiratory tract [46, 57]. Little is known about the virulence factors of these bacteria. A. haemolyticum produces phospholipase D and neuraminidase. Phospholipase D breaks down sphingomyelin by causing necrosis of the skin and subcutaneous tissue and allows the bacteria to spread into the tissues. Phospholipase D also recognises lipid compounds in host cells, leading to increased bacterial adhesion to these cells . Phospholipase D enhances activity of the cholesterol-dependent cytolysin called arcanolysin produced by the protein of this species. In the chemical structure of this enzyme, alanine occurs instead of cysteine. Arcanolysin displays specificity for human, rabbit, sheep and bovine erythrocytes . A. haemolyticum is often described as a “mysterious bacterium” because it can be considered as an impurity or microbiota component and omitted as the etiological agent of the infection [40, 57]. The most common form of A. haemolyticum infection is pharyngitis and tonsillitis. It is estimated that even 0.5–2.5% of the cases of pharyngitis can be induced by this species, and 20–50% of the cases of pharyngitis with this aetiology are accompanied by a rash [32, 40]. Less often, these bacteria can cause osteoarthritis [5, 50], necrotizing fasciitis [44, 50], dermatitis, inflammation of the soft tissue and connective tissue [23, 36]. These bacteria usually remain sensitive to antibiotics such as penicillins, cephalosporins, carbapenems, macrolides, fluoroquinolones, tetracyclines, rifampicin and vancomycin. This is confirmed by research results (unpublished data, Sękowska). In the treatment of deep infections, penicillin is used in high doses as a monotherapy or in combination with gentamicin. In our hospital, A. haemolyticum strains have been isolated mainly from diabetic patients with skin and subcutaneous tissue infections, lower limb ulcers, as well as from a stump after limb amputation.
Brevibacterium spp. is a catalase positive, obligate aerobe, able to grow in a wide range of pH (5.5–9.5) and on a substrate containing sodium chloride in a concentration of 6.5% (w/v). In the cell wall of these bacteria, the following fatty acids are present: pentadecanoic, heptadecanoic and meso-diaminopimelic. Brevibacterium spp. produces methanethiol (methyl mercaptan) from L-methionine, which determines its characteristic odour. This genus is commonly found in soil and water, as well as on human skin. It has also been isolated from fresh milk and ripe cheese, and due to its specific smell it is used in the cheese-making industry for the production of Liverat, Reclette, Limburger and Năsal cheeses. The most frequently reported cases of infections with Brevibacterium casei and Brevibacterium otitidis are associated with peritonitis [3, 11, 19, 34, 39]. The following cases of infection have been described: blood [6, 34], brain abscesses , eyeball  or central nervous system inflammation [16, 28]. In our hospital, Brevibacterium spp. strains have been isolated from the blood and biomaterials from patients with a generalised infection and catheter infection. One case of isolating B. casei from kidney preservation fluid has also been recorded, but most likely it was the contamination of the sample from the external environment. All cultured strains were sensitive to imipenem and vancomycin. In turn, the sensitivity to penicillin, cefotaxime, clindamycin and ciprofloxacin varied, from strains fully sensitive through reduced sensitivity to completely resistant strains.
Streptococci with special nutritional requirements
Other bacteria which are increasingly often isolated from clinical specimens are streptococci with special nutritional requirements, belonging to the genera Granulicatella and Abiotrophia. These bacteria require pyridoxal, cysteine and vitamin B6 to grow. They are catalase-negative, facultatively anaerobic. They are part of the upper respiratory tract microbiota, the gastrointestinal tract and the urogenital system. Depending on the availability of the aforementioned compounds in the environment, they may create forms from typical seed grains, through grain-sticks to regular cylindrical forms with a tendency to form chains. Colony morphology on solid substrates also depends on the availability of nutrients in the substrate. Around the colony, there may occur haemolysis of the type α or γ.
The following species are usually isolated from the cases of infection: G. adiacens, G. elegans, G. paraadiacens. Risk factors for Granulicatella spp. include: steroid therapy, intravenous therapy, eating disorders (including anorexia, bulimia, but also dietary change), circulatory disorders and diabetes. Most often, these bacteria cause infective endocarditis (IE) on natural and artificial valves. Granulicatella spp. may be an etiological factor even in 5% of IE [1, 37, 55, 56]. The incidence of IE with this aetiology is 44% on the aortic valve, 38% mitral, 13% tricuspid, and in 13% it is a multivalve IE [43, 48, 56]. G. adiacens, besides Streptococcus mutans and Veilonella atypica, is the most frequently isolated microorganism from the biofilm formed on prostheses . Bacteria of the Granulicatella genus may cause up to 2% of bacteraemia, and also infections of the eyes [27, 53], osteoarthritis , boils . The results of our studies confirm the participation of Granulicatella spp. in wound infections, abdominal cavities and abscesses (unpublished data, Sękowska). The isolated Granulicatella spp. strains were mostly multi-susceptible, but the strain was also cultured with sensitivity preserved only to cefotaxime. Results of the research conducted by Prasidthrathsint and Fisher  suggest that sensitivity to penicillin and ceftriaxone may be related to the species. The strains of G. elegans were sensitive to the above-mentioned antibiotics in 80% and 90% respectively, and G. adiacens in 39.4% and 47.2%. Over 90% of the strains from both species were sensitive to meropenem, levofloxacin and vancomycin. Renz et al.  described the relationship between the presence of Granulicatella spp. and the development of caries and periodontal diseases.
Abiotrophia spp., in contrast to Granulicatella spp. produces β- and α-galactosidase. The genus contains one species of Abiotrophia defectiva, which can cause bacteraemia, keratitis , oral mucositis and gingivitis , urinary tract infection  and infective endocarditis [15, 17]. More commonly, Abiotrophia spp. infections are reported in the group of patients with neutropenia. In the treatment of IE with this aetiology, the medications of choice are penicillins in combination with gentamicin . These strains remain sensitive to ceftriaxone, clindamycin, levofloxacin and vancomycin .
Another group of bacteria, in which systematic changes has taken place and new species have appeared, is Gram-negative rods from the genus Pantoea belonging to the Erwiniaceae family . The species Pantoea agglomerans has been reclassified from the genus Enterobacter. It is a flagellated bacterium which produces a yellow dye, capable of growth at 40°C. Often referred to as “agricultural bacterium”, due to its widespread occurrence in the natural environment (fruits, vegetables, plants, water, soil). It has also been isolated from animal waste. An important group with risk of infection caused by these rods is gardeners, and in hospital conditions preterm babies, new-borns with congenital malformations, as well as patients for whom biomaterials have been used. Most often a disruption of skin continuity constitutes a portal of infection , and the source of infections may be preparations administered parenterally, blood products, blood substitutes, bandages and dressings. The available literature suggests that infections with these bacteria most often affect blood [4, 8, 10, 29, 49, 54] and the respiratory system [4, 8, 14, 54]. Cases of peritonitis , endophthalmitis  and postoperative meningitis have also been reported . In the studies, Pantoea spp. strains have been isolated from wounds, blood, biomaterials and urine mainly from oncological and diabetic patients. Multidrug resistant strains have also been isolated from the body cavity fluid from a patient with acute respiratory failure and an ESBL-positive strain from a child with leukaemia. Several strains from the hospital environment (a surgical trolley and a basin siphon) have been recorded (unpublished data, Sękowska).
In the group of Gram-negative non-fermenting rods in the family Comamonadaceae, a new genus Delftia has been formed, which includes the species Delftia acidovorans (formerly Comamonas acidovorans). Reclassification was based on the 16S rRNA sequence . These are aerobic rods which produce oxidase and indole from tryptophan. This bacterium is commonly isolated from the hospital environment and has been considered non-pathogenic for many years. Delftibactin is a characteristic feature of this bacterium. It is a substance which neutralises gold ions, turning them into harmless golden nanoparticles. They accumulate outside the bacterial cells to form a red-coloured colloidal solution . Hence, this bacterium is often called “King Midas bacterium”. On solid substrates D. acidovorans grows in the form of golden-coloured or golden gloss colonies. This bacterium has also been isolated from thermised milk and sewage. Among risk factors, the presence of biomaterials and diseases: AIDS and cancer are listed . Due to the ability to create biofilms, cases of catheter-associated bacteraemia associated are most frequently reported [12, 20, 24, 30, 42, 52]. This is also confirmed by observations from our centre, although the most common strains of Delftia spp. have been isolated from swabs from chronic wounds and from surgical site infections (unpublished data, Sękowska). Descriptions of the cases of empyema , pneumonia , IE  and urinary tract infections have also appeared . The rods of D. acidovorans are susceptible to a wide range of cephalosporins, ureidopenicillin, cotrimoxazole, fluoroquinolones and tetracycline [35, unpublished data, Sękowska].
Increasingly frequent isolation of bacterial species described so far as “non-pathogenic” from infections is also associated with the growing group of immunocompromised people (angiopathy, reduced T-cell activity in the immune response, decreased neutrophil function, low level of proinflammatory cytokine secretion), civilisation or wasting diseases. On the other hand, the bacteria themselves with their easy adaptation to new living conditions also have an impact on this situation. They are often species growing in a wide range of temperatures and pH, as well as having low nutritional requirements (oligotrophy). The virulence of these bacteria is more strongly expressed in the production of factors that facilitate adhesion to host cells, colonisation of the gastrointestinal tract in particular and possibly subsequent infection.
The progress of medicine in recent years has undoubtedly positively influenced the life expectancy of patients with certain diseases (diabetes, atherosclerosis, cardiovascular diseases) and in many situations contributed to the improvement of its quality. Unfortunately, it has also made possible infections caused by bacteria, which were previously considered to be non-pathogenic, and the number of such cases will probably still rise.
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