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Abstract

The term 'unculturable' is used to describe bacteria that are not grown on artificial media till date and thus these are referred as unculturables. We do not have sufficient biological information to culture these bacteria in vitro. These microscopic cells are considered to be dead and therefore would never grow posed the anomaly without classifying them. In fact, many of these cells were shown to be metabolically active, even though they were not able to grow on laboratory media.
350 IJSR - INTERNATIONAL JOURNAL OF SCIENTIFIC RESEARCH
Volume : 2 | Issue : 9 | September 2013 • ISSN No 2277 - 8179 Research Paper
Microbiology
Dharmesh Harwani
Department of Microbiology, Maharaja Ganga Singh University, NH-15, Bikaner
334001, INDIA
ABSTRACT 

in vitro. These microscopic cells are considered to be dead and therefore would never grow posed the anomaly without classifying
them. In fact, many of these cells were shown to be metabolically active, even though they were not able to grow on laboratory media.
The Great Plate Count Anomaly and the
Unculturable Bacteria
KEYWORDS : Anomaly, Plate count,
Unculturable bacteria
THE GREAT PLATE COUNT ANOMALY
The great plate count anomaly is the observation that most of
the microbes seen in the microscope cannot currently be grown
under laboratory conditions, some may actually be nonviable,
others are viable but nonculturable (VBNC). The term “the great
plate count anomaly” was coined by Staley and Konopka (1985)
to describe the difference between the numbers of cells from
natural environments that form viable colonies on agar me-
dium and the numbers obtained by microscopy (Fig. 1). There
are several explanations for this great plate count anomaly. For
example, species that would otherwise be “culturable” may fail
to grow because their growth state in nature, such as dormancy,
prevents adjustment to conditions found in laboratory where
nutrient rich media are used for the plate counts (Deming &
Baross, 2000). If an organism has a low prevalence or is par-
ticularly slow growing it is highly likely that it may have been
over looked in the laboratory cultural analyses. Many geneti-
cally distinct phenotypes are phenotypically indistinguishable
for example some bacteria are resistant to culture on conven-
tional media, certain bacteria have fastidious growth require-
       
pH conditions, incubation temperatures or levels of oxygen in
the atmosphere (Kopke et al. 2005).
Figure 1. The Great Plate Count Anomaly
Many microorganisms are oligotrophic in nature and require
fastidious conditions for successful culture. Further there may
be competition for nutrients among mixtures of organisms cul-
tured together. Growth may also be inhibited by bacteriocins
released from other bacteria in a mixed culture or by antibacte-
rial substances present within the medium (Tamaki et al. 2005).
          
each other on a surface. This includes quorum sensing mecha-
nism that is involved in the regulation of the bacterial commu-
nity structure; properties and survival (De Kievit et al. 2001).
Signaling molecules present only within the natural habitat are
thought to be essential for the growth of many bacteria. In the
Discrete B acterial
Colonies
Dilution Plating
Environmental Sample
Microscopy
~99% bacteria
Un-culturable
Nutrient A gar Plate
Only ~ 1% Bacteria
Culturable
    -

an unfamiliar environment devoid of essential factors (Nichols
et al. 2008). Many of microbial strains that are common in na-
ture can only be cultured by specialized techniques (Baxter &
Sieburth 1984, Boogerd et al. 1989, DeBruyn et al. 1990, Koops
& Moller 1992, Ferris et al. 1996, Nold et al. 1996, Partensky et
al. 1996, Schut et al. 1997, Button et al. 1998, Vancanneyt et al.
2001, Wirsen et al. 2002).
THE EVIDENCE FOR THE UNCULTURED
BACTERIA
The evidence for the presence of uncultured bacteria that can-
not be grown in the laboratory came from molecular data. The
ability to obtain DNA sequence information from an environ-
mental sample (regardless of their viability in laboratory con-
       

such as 16S rRNA gene sequences (Amann et al. 1995). Such
sequence information uncovered a hidden treasure of bacterial
diversity that had never been acknowledged by routine culti-
vation. Woese described 11 bacterial phyla in 1987 which has
been grown now to at least 85 divisions, majority of which have
no cultured representatives (Woese 1987, Rappe & Giovannoni
2003, Keller & Zengler 2004, Achtman & Wagner 2008). The
rapid appearance of members of the uncultured phyla indicates
their presence in an environment. For example the TM7 phylum
has been detected frequently in many different environments. A

discovered in peat bogs (Rheims et al. 1996) and it has been
reported to be present in a diverse ecological conditions which
include soil, water, waste treatment sludge, marine sponges and
the human micro-biome (Hugenholtz et al. 2001, Hardoim et al.
2009, Bik et al. 2010, Dinis et al. 2011).
CATEGORIES OF THE UNCULTURABLE
BACTERIA
     -
sitic bacteria. These bacteria expand under the host provided
        Prochloron
didemni, Cristispira, Holospora, Caedibacter, Lyticum, Blattabac-
terium and rickettsiae. In the second category, viable but non
culturable organisms have been included. Though these organ-
isms are viable in natural conditions but fail to undergo cell
division on routinely used growth media. Cells of many native
marine bacteria have been tested by a number of methods to
address particularly these issues which have been observed to
    
(Colwell et al. 1985).
CONCLUSION AND FUTURE
PERSPECTIVE
Microbial diversity analysis of unculturable bacteria has re-
vealed previously uncharacterized members in bacterial do-
mains. These novel unculturable bacteria represent an un-
explored and unexploited vast gene pool. Genomic library
construction of unculturable members of various bacterial
         -
ery. Availability of community genome sequences will help in
IJSR - INTERNATIONAL JOURNAL OF SCIENTIFIC RESEARCH 351
Volume : 2 | Issue : 9 | September 2013 • ISSN No 2277 - 8179
Research Paper
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     
Bacterial evolution. Microbiology Reviews, 51:221–271.
In addition to that an
access to the neglected bacteria that reside on us may provide
new avenues to improve overall health through an enhanced
understanding of the yet unknown functionalities. Although ad-
vances in recent research have shown that members of bacterial
groups that were previously considered to be unculturable can
now be cultured, a major part of the existing bacterial diversity
still remains cryptic due to their culture recalcitrance.
... Unculturable bacteria have a wide variety. Some are resistant to culture using synthetic media, some have fastidious growth requirements, and some need specific physical conditions to survive, such as pH, temperature, and oxygen levels [19,20]. A few categories of unculturable bacteria include obligatory symbiotic and parasitic bacteria, which proliferate under conditions set by the host but not on artificial media, and viable but non-culturable bacteria, which are viable in the natural environment but unable to divide in regularly used growth mediums [19,21]. ...
... Some are resistant to culture using synthetic media, some have fastidious growth requirements, and some need specific physical conditions to survive, such as pH, temperature, and oxygen levels [19,20]. A few categories of unculturable bacteria include obligatory symbiotic and parasitic bacteria, which proliferate under conditions set by the host but not on artificial media, and viable but non-culturable bacteria, which are viable in the natural environment but unable to divide in regularly used growth mediums [19,21]. Marine bacteria are also known for being unculturable due to the existence of dormant cells, low growth rates, poor colony development, the need for metabolites produced by other microorganisms, and inadequate growth conditions [22]. ...
... Past efforts to identify microorganisms in oil reservoirs were mostly based on cultivation in growth media [10][11][12] . However, the unculturable portion of environmental microbes is around 90%-99% 13 and it is reasonable to assume that this holds true for oil reservoirs as well 14 . To understand microbial reservoir dynamics, it is imperative to identify the taxa with cultivation-independent methods. ...
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Microbes from oil reservoirs shape petroleum composition through processes such as biodegradation or souring. Such processes are considered economically detrimental and might pose health and safety hazards. It is therefore crucial to understand the composition of a reservoir's microbial community and its metabolic capabilities. However, such analyses are hindered by difficulties in extracting DNA from such complex fluids as crude oil. Here, we present a novel DNA extraction method from oils with a wide American Petroleum Institute (API) gravity (density) range. We investigated the ability to extract cells from oils with different solvents and surfactants, the latter both nonionic and ionic. Furthermore, we evaluated three DNA extraction methods. Overall, the best DNA yields and the highest number of 16S rRNA reads were achieved with isooctane as a solvent, followed by an ionic surfactant treatment using sodium dodecyl sulfate and DNA extraction using the PowerSoil Pro Kit (Qiagen). The final method was then applied to various oils from oil reservoirs collected in aseptic conditions. Despite the expected low cell density of 10 1-10 3 cells/ml, the new method yielded reliable results, with average 16S rRNA sequencing reads in the order of 41431 (±8860) per sample. Thermophilic, halophilic, and anaerobic taxa, which are most likely to be indigenous to the oil reservoir, were found in all samples. API gravity and DNA yield, despite the sufficient DNA obtained, did not show a correlation.
... Heterotrophic bacteria colony growth was ultimately used in ARG analysis and provided an estimate of bacteria load, or the number of viable bacteria in a system. The heterotrophic bacteria concentrations are a conservative estimate of viable bacteria load as many bacteria have stringent temperature, nutrient, and atmospheric conditions and will not grow on media or in a laboratory environment (Harwani, 2012;Köpke et al., 2005). Although assessing ARGs in heterotrophic bacteria growth is an underestimate of an environment's resistome, it does provide critical insight into baseline ARG signatures and highlights specific sites, environmental variables, and watershed characteristics related to antibiotic resistance potential. ...
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Antimicrobial resistance is now recognized as a leading global threat to human health. Nevertheless, there currently is a limited understanding of the environment's role in the spread of AMR and antibiotic resistant genes (ARG). In 2019, the U.S. Geological Survey conducted the first statewide assessment of antibiotic resistant bacteria (ARB) and ARGs in surface water and bed sediment collected from 34 stream locations across Iowa. Environmental samples were analyzed for a suite of 29 antibiotics and plated on selective media for 15 types of bacteria growth; DNA was extracted from culture growth and used in downstream polymerase chain reaction (PCR) assays for the detection of 24 ARGs. ARGs encoding resistance to antibiotics of clinical importance to human health and disease prevention were prioritized as their presence in stream systems has the potential for environmental significance. Total coliforms, Escherichia coli (E. coli), and staphylococci were nearly ubiquitous in both stream water and stream bed sediment samples, with enterococci present in 97 % of water samples, and Salmonella spp. growth present in 94 % and 67 % of water and bed sediment samples. Bacteria enumerations indicate that high bacteria loads are common in Iowa's streams, with 23 (68 %) streams exceeding state guidelines for primary contact for E. coli in recreational waters and 6 (18 %) streams exceeding the secondary contact advisory level. Although antibiotic-resistant E. coli growth was detected from 40 % of water samples, vancomycin-resistant enterococci (VRE) and penicillinase-resistant Staphylococcus aureus (MRSA) colony growth was detected from nearly all water samples. A total of 14 different ARGs were detected from viable bacteria cells from 30 Iowa streams (88 %, n = 34). Study results provide the first baseline understanding of the prevalence of ARB and ARGs throughout Iowa's waterways and health risk potential for humans, wildlife, and livestock using these waterways for drinking, irrigating, or recreating.
... The history of metagenomics can be dated back to the report of the "great plate count anomaly", which evidenced that cultured microorganisms cannot represent the whole microorganism world [1]. Then, after the notion emerged that 16S rRNA genes can be used as a marker for differentiating between bacterial species, environmental DNA was directly cloned from the ocean for the 16S rRNA gene sequence analysis [2]. ...
Article
Full-text available
The development of metagenomics has opened up a new era in the study of marine microbiota, which play important roles in biogeochemical cycles. In recent years, the global ocean sampling expeditions have spurred this research field toward a deeper understanding of the microbial diversities and functions spanning various lifestyles, planktonic (free-living) or sessile (biofilm-associated). In this review, we deliver a comprehensive summary of marine microbiome datasets generated in global ocean expeditions conducted over the last 20 years, including the Sorcerer II GOS Expedition, the Tara Oceans project, the bioGEOTRACES project, the Micro B3 project, the Bio-GO-SHIP project, and the Marine Biofilms. These datasets have revealed unprecedented insights into the microscopic life in our oceans and led to the publication of world-leading research. We also note the progress of metatranscriptomics and metaproteomics, which are confined to local marine microbiota. Furthermore, approaches to transforming the global ocean microbiome datasets are highlighted, and the state-of-the-art techniques that can be combined with data analyses, which can present fresh perspectives on marine molecular ecology and microbiology, are proposed.
... Although there are many bacteria in soil (1,2) a large number of them are unculturable (3,4). Known as the "Great Plate Count Anomaly" (5), if a sample is observed directly from the soil using microscopy, it will have more cells than a sample grown on a petri plate in terms of colony forming units (CFUs) (6). Thus this causes the "Great Plate Count Anomaly" due to the discrepancy in direct cell counts of bacteria in comparison to counts based on culturing bacteria. ...
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Full-text available
In this study, we aim to identify bacterial composition in soils treated with and without wood chips, as well as high levels and low levels of nitrogen. In order to do this, we used both culture dependent methods including plating, observing, and GenIII plates; as well as culture independent methods which include microbiome sequencing and EcoPlates. By doing this, we concluded that the bacterial communities differ between unamended and wood-chipped treated soils. Multiple differences were identified: rate of carbon consumption, bacterial diversity, rRNA gene copy number, and colony morphologies. Identifying the bacteria in wood-chipped soils will allow us to understand their effect on biogeochemical cycles, plant pathogens, and relationship with the trees (symbiotic/pathogen). Additionally, understanding the effect lower nitrogen levels have on the bacterial composition of soils can lead to reduction of synthetic fertilizer use, therefore lessening the environmental impact of nitrogen.
... It is possible that this discrepancy reflects biases in our metagenome inference software (45), as the Nearest Sequence Taxon ID (NSTI) values for our samples indicated relatively low representation of many of our taxa in published databases, although our pipeline removed highly divergent OTUs from the metagenome inference to minimize this problem. A more likely cause is that culture-based assays of soil communities are potentially misleading due to the strong cultivation bias in these systems (46). The great majority of our isolates fell into a few clades that were not closely related to the most abundant taxa from our tag sequencing analysis (Fig. S6), and it is noteworthy that only 3 of the 48 OTUs making up more than 1% of any sample (Bacillus, Rhizobium, and Streptomyces) had a close relative among the isolates. ...
Article
Full-text available
Heavy metals (HMs) are known to modify bacterial communities both in the laboratory and in situ. Consequently, soils in HM-contaminated sites such as the U.S. Environmental Protection Agency (EPA) Superfund sites are predicted to have altered ecosystem functioning, with potential ramifications for the health of organisms, including humans, that live nearby. Further, several studies have shown that heavy metal-resistant (HMR) bacteria often also display antimicrobial resistance (AMR), and therefore HM-contaminated soils could potentially act as reservoirs that could disseminate AMR genes into human-associated pathogenic bacteria. To explore this possibility, topsoil samples were collected from six public locations in the zip code 35207 (the home of the North Birmingham 35th Avenue Superfund Site) and in six public areas in the neighboring zip code, 35214. 35027 soils had significantly elevated levels of the HMs As, Mn, Pb, and Zn, and sequencing of the V4 region of the bacterial 16S rRNA gene revealed that elevated HM concentrations correlated with reduced microbial diversity and altered community structure. While there was no difference between zip codes in the proportion of total culturable HMR bacteria, bacterial isolates with HMR almost always also exhibited AMR. Metagenomes inferred using PICRUSt2 also predicted significantly higher mean relative frequencies in 35207 for several AMR genes related to both specific and broad-spectrum AMR phenotypes. Together, these results support the hypothesis that chronic HM pollution alters the soil bacterial community structure in ecologically meaningful ways and may also select for bacteria with increased potential to contribute to AMR in human disease. IMPORTANCE Heavy metals cross-select for antimicrobial resistance in laboratory experiments, but few studies have documented this effect in polluted soils. Moreover, despite decades of awareness of heavy metal contamination at the EPA Superfund site in North Birmingham, Alabama, this is the first analysis of the impact of this pollution on the soil microbiome. Specifically, this work advances the understanding of the relationship between heavy metals, microbial diversity, and patterns of antibiotic resistance in North Birmingham soils. Our results suggest that polluted soils carry a risk of increased exposure to antibiotic-resistant infections in addition to the direct health consequences of heavy metals. Our work provides important information relevant to both political and scientific efforts to advance environmental justice for the communities that call Superfund neighborhoods home.
... The validity and reasoning behind the calculation and implications are much discussed in the literature (e.g. [71]). ...
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Full-text available
Collectively, we have been reviewers for microbial ecology, genetics and genomics studies that include environmental DNA (eDNA), microbiome studies, and whole bacterial genome biology for Microbial Ecology and other journals for about three decades. Here, we wish to point out trends and point to areas of study that readers, especially those moving into the next generation of microbial ecology research, might learn and consider. In this communication, we are not saying the work currently being accomplished in microbial ecology and restoration biology is inadequate. What we are saying is that a significant milestone in microbial ecology has been reached, and approaches that may have been overlooked or were unable to be completed before should be reconsidered in moving forward into a new more ecological era where restoration of the ecological trajectory of systems has become critical. It is our hope that this introduction, along with the papers that make up this special issue, will address the sense of immediacy and focus needed to move into the next generation of microbial ecology study.
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Diatoms are among the most diverse and environmentally significant protists on Earth. Like many other organismal groups, a large portion of their diversity appears to lie beyond the resolution of the traditional light microscopy-based methods routinely and sometimes exclusively utilized in their investigation. Although the technological and conceptual developments in the fields of molecular biology and bioinformatics unlocked a remarkable opportunity to study diatoms in a previously unimagined depth and breadth, molecular diatomists anecdotally claim that diatoms remain genetically understudied compared to other taxa. However, this claim has never been quantified and rigorously tested. Therefore, we performed a bibliometric analysis of over 42,000 WoS-indexed diatom documents published in the past 35 years, between 1988 and 2023. The claim is confirmed: only ~15% of the analyzed diatom literature incorporated molecular data, about half compared to other groups, including other algae, cyanobacteria, plants, fungi, and animals. Interestingly, research for all groups seems to asymptotically saturate with molecular methods once they are used in about one-third of the documents annually, an observation which has important implications. In addition, past trends in the use of molecular data in diatomology were explored and some future ones were predicted.
Article
Viruses shape microbial community structure and activity through the control of population diversity and cell abundances. Identifying and monitoring the dynamics of specific virus-host pairs in nature is hampered by the limitations of culture-independent approaches such as metagenomics, which do not always provide strain-level resolution, and culture-based analyses, which eliminate the ecological background and in-situ interactions. Here, we have explored the interaction of a specific “autochthonous” host strain and its viruses within a natural community. Bacterium Salinibacter ruber strain M8 was spiked into its environment of isolation, a crystallizer pond from a coastal saltern, and the viral and cellular communities were monitored for one month using culture, metagenomics, and microscopy. Metagenome sequencing indicated that the M8 abundance decreased sharply after being added to the pond, likely due to forces other than viral predation. However, the presence of M8 selected for two species of a new viral genus, Phoenicisalinivirus, for which 120 strains were isolated. During this experiment, an assemblage of closely related viral genomic variants was replaced by a single population with the ability to infect M8, a scenario which was compatible with the selection of a genomic variant from the rare biosphere. Further analysis implicated a viral genomic region putatively coding for a tail fiber protein to be responsible for M8 specificity. Our results indicate that low abundance viral genotypes provide a viral seed bank that allows for a highly specialized virus-host response within a complex ecological background.
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