Methylobacterium spp. - ubiquitous bacterium, plant growth promoter and human opportunistic pathogen: qualitative and quantitative culture, molecular diagnosis (PCR) and species identification (PCR and sequencing)

Methylobacteria were bacteria that before being included in the genus Methylobacterium created in 1976, were included in other genera such as Pseudomonas. They are coccobacillary bacteria, which usually appear grouped or sometimes forming chains, gram-negative, vacuolated, strict aerobic, most of them mobile by a single flagellum. Physiologically they are facultative methylotrophic, since they can use carbon sources from molecules with several carbon atoms, but they differ from other bacteria because they can develop from molecules with a single reduced carbon atom (different from CO2). Its growth is difficult and of slow development that requires 7 to 10 days to form colonies in solid culture media. Their colonies are pink pigmented, 1 to 2 mm in diameter depending on the growth time.

Being facultative methylotrophic, they can grow in culture media containing carbon compounds with molecules of a single carbon atom (without carbon bonds) such as methanol, formadehyde, formic acid and some methylated (methylamine) or halogenated amines. According to the carbon molecules that they can use, two groups have been differentiated, those that use methane as the only carbon source and those that cannot use it. This fact makes it easier for them to be found in environments with few nutrients (oligotrophic) such as natural surface waters, water reservoirs, or even drinking water distribution networks because they are resistant to chlorine in treated water.

Its optimum growth temperature is 25 to 30ºC. Metabolically they are producers of catalase, oxidase and urease, not producers of indole or H2S. Both the methyl red reaction and the Voges-Proskauer test are negative. They hydrolyze starch, but they do not hydrolyze gelatin, esculin, or DNA; they reduce nitrates, use citrate as a carbon source (Simmons citrate test), generate acid from D-arabinose, but not from D-glucose, D-galactose, D-mannose, or maltose. They do not grow in the presence of 10% NaCl. More than 34 different species have been described. However, its slow growth makes phenotypic identification through metabolic tests very difficult, so its molecular identification is more practical, either with genus amplification primers (PCR), or 16S rRNA gene sequencing for species identification.

Due to the pink pigmentation of their colonies, they can be mistaken with other bacteria whose colonies become pigmented, such as Serratia, Azospirillum, Roseomonas and Asaia.

They are saprophytic bacteria, widely distributed in nature, being frequent in fresh water and wastewater, soil, lake sediments, air, plant leaves, rice grains, cattle rumen, and even in hospital environments. In plants they are found as epiphytic colonizers in the leaves or endophytes, colonizing the germination or growth shoots intracellularly. As symbionts they are found in root nodules, where they can benefit from methanol generated by plants. They produce phytohormones that stimulate seed germination and plant growth, increasing seed germination, leaf area, or plant height (Plant growth promoting rhizobacteria –PPR-).

They have also been found related to microalgae. The increase in interest of microalgae both from an industrial point of view due to their high lipid content and their ability to accumulate high-value compounds, such as food to be used as food supplements and for animal feed, and even for the production of biofuels, or synthetic fertilizers. By associating with microalgae, they would stimulate their growth and morphogenesis by releasing essential minerals, vitamins, auxins that regulate cell growth, and molecules involved in the mechanism of “Quorum sensing”.

These bacteria show relevance from a clinical poit of view because they can form part of the body´s transient flora and cause opportunistic infections due to contamination from various environmental sources. They have been found as opportunistic pathogens when there are underlying processes, especially in immunosuppressed patients, but also in patients subjected to invasive diagnostic processes in which instruments that cannot be adequately sterilized are used. Opportunistic infections can be located in the blood (bacteremia), bone marrow, respiratory system (pneumonia, pleural empyema), peritoneum, meninges, joints or skin. These infections occur mainly in immunosuppressed patients, such as those affected by malignant neoplasms (lung, bladder, uterus, ...), lymphomas, leukemias after bone marrow transplantation, infected by human immunodeficiency virus (HIV/AIDS), kidney failure, multiple sclerosis or alcoholism.

As they are present in the water pipes of health centers, together with their resistance to chlorine, they are a source of infection in dental practices, in surgical interventions when hand washing is carried out with tap water, in units where instruments that are used cannot be adequately sterilized as flexible endoscopes that come into contact with mucous membranes (respiratory, gastrointestinal, peritoneum, genitourinary tract, ...) and that are treated with glutaraldehyde solutions up to 2% to which they are resistant.

Antimicrobial treatment requires knowing their empirical sensitivity since, due to their slow culture characteristics, they are not conducive to waiting for the results of the sensitivity tests. The most recommended antimicrobials are amikacin, gentamicin, ciprofloxacin, TMP-SMX, ceftriaxone, ceftizoxime, or imipenem.

Its difficult and slow growth in microbiological culture media can make its discovery and microbiological diagnosis difficult in laboratories since cultures are not usually maintained for the time required for their development, nor are they usually incubated at their optimal growth temperature. For this reason, molecular methods have been developed that obviate the problems of its culture and avoid false negative results in its diagnosis.

Tests carried out at IVAMI:

  • Qualitative and quantitative culture. 
  • Molecular diagnosis (PCR). 
  • Molecular identification of species (sequencing).