Advancements in methods to detect and culture medically important anaerobic bacteria

Anaerobic bacteria are one of the most important bacteria, involved in a number of diseases and infections. These are also involved in food borne illness. Due to their fastidious nature, culturing anaerobic bacteria is a bit difficult task. Moreover, anaerobic bacteria can take several days and weeks to grow in laboratories. Apart from this, most bacteria just cannot be cultured in laboratories using standard (anaerobic) cultivation techniques known so far. Difficulties in microbiological detection result in delayed diagnosis of the diseases. Many patients suffer due to these facts, as rapid identification is not only difficult, but in many cases, is almost impossible. Thus, there is a need to develop novel techniques for the cultivation and identification of clinically important anaerobes. Rapid detection of foodborne pathogens is necessary for the prevention of foodborne disease and for the safe supply of food. Present article reviews and discusses advance techniques, both culture-dependent and culture-independent, that allow rapid detection of such important anaerobic Copyright © 2021 The Authors. Production and hosting by Department of Life Sciences, University of Management and Technology is licensed under a Creative Commons Attribution 4.0 International License


Introduction
Anaerobes are the dominant members of normal human microbiota, predominantly residing on mucosal membranes of oral cavity, gastrointestinal tract, and female genital tract. Being a major component of oral microbiota, their concentration ranges from 10 2 ml -1 in saliva to 10 12 ml -1 in gingival scrapping. Gastric acidity is responsible for minimizing their number in stomach and upper intestine, however, in colon their concentration reaches up to 99% of the total bacterial burden which is 10 12 organisms per gram of stool.
Clostridium, Pepetostreptococcus, Fusobacterium and Bacteroid species are the culturable anaerobes however many of the anaerobes are found to be uncultivable by conventional laboratory techniques known so far. Breakdown of mucosal barrier can lead to the contamination of anaerobes in the sterile sites of the body leading to the severe infections Bennett et al. 1 and can cause for 1-17% positive blood cultures 2 . Moreover, they are also involved in food-born infection, lung infections and brain abscesses. According to Center for Disease Control and Prevention, 179 million people get sick due to foodborne pathogens and 6,186 die each year in United States 3 . Table 1 shows some of the important pathogenic bacteria and the diseases that they cause.
Anaerobic spore formers (clostridium and related genera) are a significant concern for foodborne illness due to toxin production.
They can be toxigenic, neurotoxigenic or spoilage bacteria. Clostridium botulinum and Clostridium tetani are neurotoxigenic species.
While Clostridium perfringens is a prominent foodborne pathogen, the second largest cause of food poisoning in the USA, where it causes nearly 1,000,000 cases per annum with net financial load of US$382 million 4,5 . It is also responsible for diarrhea, avian enteritis necroticans, fulminant disease, clostridial myonecrosis and enterotoxemia due to production of variety of toxins with diverse characteristics 6 .
Over the past 20 years, it has been reported that diagnostic difficulties can hinder the rapid detection and identification of anaerobes on a species level. Cultivation of strict anaerobes in microbiology laboratory can be challenging as it demands highly equipped systems with strict anaerobiosis and reduced culture media, owing to the fact that oxygen is toxic for most of the anaerobes. Moreover, most anaerobic species are slow growing (can take up to 14 days to grow) and many are inactive for certain biochemical tests. Beside classical methods; including selective media, evaluation of rapid tests (such as nitrate disks, spot indole, growth in 20% bile containing media, etc), commercial kits are also being used for their detection. In spite of the capability and accuracy of these commercial kits, some clinically important anaerobic pathogens such as Prevotella nanceiencis, Bacteroides nordii, Bacteroides cellulosilyticus, Bacteroides dorei and many others are commonly either misidentified not identified at all 7, 8 9 . Updated culture based as well as molecular based techniques such as 16S rDNA sequencing and QRT-PCR has enabled rapid detection and identification of many anaerobes. Various Bacteroide species have been detected from clinical samples by use of QRT-PCR 10 11 .
In this article, we review recent techniques, both culture based and molecular, that can be used to comparatively rapid detection of such important anaerobes.

Culture-dependent techniques
To culture strict anaerobes, techniques capable of generating rapidly a low oxygen level (<0.5%) atmosphere are suitable 12 .
Anaerobic chambers are being used for generation of such anaerobic atmosphere, however, it can be expensive for most laboratories.
Generation of anaerobic atmosphere by chemical compounds [such as sodium borohydride (NaBH4), sodium bicarbonate-citric acid] is more applicable as compared to bulky anaerobic chamber 13 . Such chemical compounds have been utilized in the development of a disposable Quick anaero-system for culturing the strict anaerobes. The whole system consists of three components. First one is a disposable anaerobic gas pack which was developed to maintain absolute anaerobic atmosphere. This component is further subdivided into 2 subunits. First part is equipped with silica (SiO2) and NaBH4 tablets. The other part produces CO2, generated by the reaction of sodium bicarbonate with citric acid. These two parts are connected with a narrow-tipped (10ml) plastic tube which is also used for pouring of tap water. Second component is a disposable culture envelope, a sealer and a reusable rack. Third component is catalyst unit which utilizes 10g alumina pellets coated with 0.5% palladium, kept below the roof of the rack. Working of this gas container is based on the generation of volatile hydride (SiH4), produced by the reaction of SiO2 and NaBH4 tablets with water. Efficacy of this quick anaero-system was evaluated by culturing 12 anaerobes in both the Quick anaero-system and BD GasPak EZ Anaerobe System. By comparing the growth of the anaerobes in both the systems, it was estimated that 2 out of 12 and 9 out of 12 anaerobes (cultured on LBand Blood-agar plates, respectively) showed better growth in Quick anaero-system 14 .
For the isolation of anaerobes from rumen, a new medium was used by Kenters et al. 15 to assess the concentrations of inorganic components of rumen, so that it may mimic to the chemical environment of rumen. Major components of this bicarbonate-buffered mineral media are KH2PO4, (NH4)2SO4, KCl, NaHCO3, L-cysteine·HCl·H2O, resazurin solution and trace element solution SL10. Media preparation is done in O2-free 100% CO2 atmosphere. After the collection of rumen contents from animals, rumen fluid, substrates and Vitamin 10 concentrate are prepared as described by Kenters et al. 15 . Rumen samples are prepared for the cultivation and the cells are harvested after the incubation. The method has proved to be a successful tool for the cultivation of Firmicutes, Bacteroidetes, and Spirochaetes, as confirmed by 16S rDNA comparative analysis.
Oxygen toxicity is a major problem for anaerobes which can be avoided by the use of antioxidant molecules. In a research work by La Scola et al. 16 the efficacy of antioxidant molecules was evaluated by cultivating the obligate anaerobes in aerobic atmosphere.
For all the tubes, supplemented with antioxidants, the growth was observed within 0.3cm of the surface area, but there was no growth in control plates (without ascorbic acid). Thus the use of antioxidants is proved to be a successful method for culturing these clinical anaerobic isolates in aerobic atmosphere. Moreover, this media also allows the growth of aerobic bacteria. Utilization of antioxidants can be regarded as an easy approach for cultivation of anaerobes with a regular incubator in aerobic environment 16 .
Replacement of the atmospheric oxygen with O2-free gases along with the usage of reducing agents in culture media are useful steps to further facilitate the cultivation of anaerobes. A six well plate method was developed by Nakamura et al. 17 which works together with the AnaeroPack System 18 . This method was proved to be a better technique for culturing the strict anaerobes like methanogens, sulfate reducing bacteria and hydrogen-producing syntrophs. The method was examined by inoculation of anaerobes in both aerobic and anaerobic conditions. Methanogens and sulfate reducing bacteria were used as test organisms. Sterilized W-gellan media (supplemented with reductants) was poured into each well and was inoculated with the culture dilutions (with the ratio of 13:0.1). The plates were covered with lid and kept in nylon bag equipped with two catalyst sachets (AnaeroPouch). For anaerobic inoculation, the whole method was also done in anaerobic chamber. The bag was sealed after replacing its atmosphere with H2/CO2 or N2/CO2. For comparison, role tube method was also performed as described by Hungate 19  Recent advances in polyclonal and monoclonal antibody production increased the sensitivity and specificity of immunological assays, resulting in quick identification of foodborne pathogens and toxins 24 25 . Enzyme-Linked Immunosorbent Assays (ELISA), Lateral Flow Immunoassays and Immunomagnetic Separation Assays are found to be rapid immunological techniques for detection of many foodborne pathogens such as Salmonella spp, E. coli as well as for botulinum toxins and enterotoxins 23 . Clostridium difficile infection (CDI) is one of the most important nosocomial infection, causing economic burden on health care system worldwide, and is considered as a serious public health threat by the U.S. Center for Disease Control and Prevention 12,32 .
Limitations in laboratory diagnostic capacity has made CDI identification a difficult task in developing countries. A workflow has been proposed by Cheng et al. 33 which shows 100% sensitivity and 92.8% specificity, compared to culture dependent methods. Fecal samples were collected and were tested for glutamate dehydrogenase (GDH) and C. difficile toxin A & B (CDAB) by commercial VIDAS kits (bioMérieux). GDH is produced by all strains of C. difficile. Use of these commercial kits is an automated and easy approach for rapid detection of C. difficile. Use of GDH assay for screening of the suspected CDI fecal samples is the first step of this work flow. GDH positive specimens are then tested CDAB for toxin detection. Negative CDAB results can be referred for molecular detection of toxin genes. This new workflow with a combination of different testing methods is found to be a successful technique which can improve the diagnosis rate from 8.2% to 19.2% with reasonable cost (from US$8 to US $15.6) 33 .
Microbial source tracking methods (MST) are also useful tools to track down the source of the contaminant bacteria. Use of host specific PCR is a popular approach of MST. It has been used for determination of fecal pollution, as it detects the genetic markers of fecal microbes specific to a specific host 34 .
Many commercial kits, both culture-and molecular based, are also available for rapid detection of clinically important bacteria.
Different companies have made effort to launch a variety of commercial products, which work either on culture based or molecular based approach for detection and identification of clinically important anaerobes. Some advanced commercial products including culture Media, Gas generator system, reagent and detection kits have been enlisted in table 2.
This will help lessen the sufferings of the patients along with reduction in mortality rates.

Conclusion
It is evident that the rapid identification of the pathogens is crucial for the timely treatment of the patients and can prove lifesaving. With ever growing illness cases, especially with the spread of multi-drug resistance, it is of utmost concern that more rapid and efficient techniques to culture and detect anaerobes be introduced. Developments in scientific techniques can help reduce detection time of anaerobic pathogens. Likewise, molecular techniques can also be tweaked for the rapid detection of the infectious agents. Moreover, to culture the unculturable pathogens, genomic studies can be helpful to find out their culture requirements.