by Prof. Devika de Costa

(Chair Professor of Plant Protection, Faculty of Agriculture, University of Peradeniya)

Background

The bulk carrier, Hippo Spirit, carrying an organic fertiliser shipment of 20,000 metric tons manufactured by the Qingdao Seawin Biotech Group Co. Ltd., China,started to sail to its destination, Colombo, Sri Lanka, from Qingdao port, China, on the 22 September 2021. Seventy-two days after its departure, on the 04 December 2021, Hippo Spirit left the Sri Lankan waters heading towards Singapore without unloading its shipment of organic fertiliser at the planned destination.

The reason for not allowing the shipment to be unloaded by the Sri Lankan authorities was the detection of the contamination of the organic fertiliser onboard with a plant pathogenic bacterium named Erwinia spp. and another group of bacteria of Bacillus spp., as confirmed twice by the National Plant Quarantine Service, Sri Lanka (NPQS). As per the Plant Protection Act No. 35, 1999 of Sri Lanka, material containing any organism harmful to, or injurious to, or destructive of plants and for the sanitation of plants in Sri Lanka, is not allowed to be imported.

Qingdao Seawin Biotech Group Co. Ltd., the supplier, directed the Hippo Spirit, along with the bulk organic fertiliser shipment, to a test laboratory in Singapore, with the intention of getting a sample of the shipment tested for its quality by a third party and to initiate an international arbitration procedure.

The third party testing laboratory that the Qingdao Seawin selected was SGS Testing & Control Services Singapore Pvt. Ltd. (SGS). The reason for obtaining the services of a third party for the testing procedure was to support the allegations made by Qingdao Seawin against the NPQS. Qingdao Seawin denied the fact that their shipment was contaminated with the bacterium Erwinia, emphasising that NPQS had not conducted the test procedures scientifically and had not followed standard methodologies adopted by the International Plant Protection Convention (IPPC).

Accordingly, a sample of the organic fertiliser shipment in Hippo Spirit was submitted to the SGS laboratory for testing its microbial quality on the 13 December 2021. At the SGS laboratory, this sample was tested to detect the presence of a specific group of bacteria, according to a standard test procedure, and the test results were released on 20 December 2021.

SGS test report Figure 1

shows an image of the test report released by the SGS laboratory on the 20 December 2021. According to the report, the submitted sample was tested by two standard methods, namely ISO 21528-1 (2017) and ISO 21528-2 (2017), to detect bacteria belonging to the Family Enterobacteriaceae, a sub-group within the large taxonomic group of bacteria.

The testing period of the sample has been reported as seven days from the 13th to the 20 December 2021. Results released by the SGS laboratory reveal that bacteria belonging to Enterobacteriaceae have not been detected in the sample subjected to the above two testing procedures.

(see Figure 1: The SGS test report)

What are Enterobacteriaceae bacteria?

Enterobacteriaceae is a large family of bacteria consisting of 53 bacterial genera (Reference No. 1). According to ‘Gram Staining’, the standard staining technique used in bacteriology for initial categorisation of bacteria, the bacteria belonging to this Enterobacteriaceae are classified as ‘Gram negative’. A majority of the bacterial genera belonging to this family are inhabitants of the human and animal intestine. Such bacteria are termed as ‘enteric bacteria’. In addition to the enteric bacteria, some of the genera belonging to the family Enterobacteriaceae live in natural environments such as soil and water.

There are some bacterial genera of this family, which are pathogenic (i.e. having the ability to cause diseases) to plants and animals. Most of the bacterial members of this family grow well at a temperature of 37 oC but there are some bacteria that show a better growth at a temperature range of 25 – 30 oC. Therefore, it is clear that within the same bacterial family, there are different bacterial genera and species with different physical, nutritional and functional characteristics. It is equivalent to siblings of a given family having differences in terms of their external morphological features, behaviour, attitudes, performances, working efficiency, etc.

Scientific basis of the test protocol to determine the quality of the sample

ISO 21528-1 (2017) and ISO 21528-2 (2017) are recommended test protocols to detect the presence of bacteria belonging to the Family Enterobacteriaceae in food samples used for human/animal consumption or to test the environmental samples used for primary food production, food production and food handling. Using the above test protocols it is possible to detect the presence of ‘culturable bacteria’ (i.e. bacteria which are capable of growing on synthetic culture media) belonging to the family Enterobacteriaceae. This means that the selected test protocols are suitable for detecting only a limited, focused group of bacteria and that these specific test protocols have not targeted the detection of all types of bacteria. The specificity of this test protocol to target only the culturable type bacteria of the Enterobacteriaceae family has been achieved through the specific synthetic culture medium used in the protocol. The culture medium used here is called the ‘Violet Red Bile Glucose Agar medium (VRBGA)’. Microbiologically, the culture media which specifically target a particular group of bacteria or microorganisms are termed ‘selective media’. Such selective media favour the growth of a particular group of microorganisms while suppressing the growth of all other types.

Selectivity of the VRBGA medium is determined by the ingredients used when preparing it. For example, the chemical stain called crystal violet and bile salts are used when preparing the VRBGA medium and both these chemical ingredients suppress the growth of Gram positive type bacteria. Moreover, the bile salts used in this medium encourages the growth of bacteria that are resistant to bile salts. In other words, only the bacteria which are not destroyed by the action of bile salts will grow on the VRBGA medium.

On the other hand, the bacteria which cannot tolerate bile salts will not grow on this selective medium even if they are present in the test sample.

Enteric bacteria naturally inhabit in an environment containing bile salts (i.e. the gut of animals and humans) and consequently are usually resistant to bile salts. Therefore, growth of such bacteria will not be suppressed when a test sample is allowed to grow on the VRBGA medium. However, non-enteric bacteria usually live in environments such as soil or plant tissue, and therefore, have no exposure to bile salts. Accordingly, they are not accustomed to grow in an environment containing bile salts. As a consequence, the growth of non-enteric bacteria is suppressed on the VRBGA medium. Therefore, it is clear that a test protocol using the VRBGA medium is not appropriate to test the presence of microorganisms or bacteria in a test sample of plant origin such as the organic fertilizer consignment from Qingdao Seawin Biotech Group Co. Ltd. More importantly, this renders the conclusion by the SGS test report invalid.

Erwinia spp. grow naturally in plant tissues which have no bile salts in them. Thus it is a group of non-enteric bacteria. As explained above, the growth of such non-enteric bacteria is heavily suppressed on the VRBGA medium. According to the results of the publication given as Reference Number 2, the VRBGA medium has been identified as a medium which underestimates the presence of plant bacteria such as Erwinia, despite Erwinia being a member of the Family Enterobacteriaceae. Therefore, it should be clear that even if bacteria belonging to Erwinia spp. had been present in the tested organic fertilizer sample from Qingdao, there was a very high probability for them not being able to develop colonies on the VRBGA medium. Thus, the conclusion that the sample is free from Erwinia is erroneous and inconclusive.

Scientific reasons for not agreeing with the conclusions made by the SGS report

The SGS report concludes that based on ISO 21528-1 (2017) and ISO 21528-2 (2017) test protocols, the sample does not contain bacteria belonging to the family Enterobacteriaceae, and therefore, the sample is free from Erwinia as well. Based on the above test report, news reports were released through public media (Daily News, 24th of December 2021 and News First, 22nd of December 2021) saying that the organic fertiliser shipment was free from plant, animal and human pathogens including Erwinia.

Given below are the scientific reasons to emphasise that the conclusion drawn is erroneous:

(a) Because of the highly selective nature of the VRBGA medium, all bacteria in the Enterobacteriaceae family will not develop colonies on it when the sample is tested by the above ISO procedures. Accordingly, Non-enteric bacteria and bacteria which are usual inhabitants of bile salt-free environments, and hence are not resistant to the effect of bile salts will not develop on the culture medium even if they are present in the test sample. Therefore, it is erroneous to conclude that the sample is free from Erwinia;

(b)

As a selective medium, VRBGA targets the detection of bacteria belonging to the Enterobacteriaceae family only. This medium suppresses the growth of all Gram positive type bacteria and other non-Enterobacteriaceae bacteria. There are many plant, animal and human pathogenic bacteria which belong to many other taxonomic families (i.e. other than Enterobacteriaceae/non-Enterobacteriaceae). Therefore, based on this test report, it is not possible to conclude that the sample is free from plant, animal and human pathogens from taxonomic families other than Enterobacteriaceae;

(c)

In giving their conclusion, the SGS report assumes that Erwinia belongs to the family Entereobacteriaceae. However, according to the most recent taxonomic classification of bacteria as given in Reference No. 3, the genus Erwinia is no longer a member of the Family Enterobacteriaceae. Accordingly, Erwinia genus is now included in a separate family named Erwiniaceae. This recent taxonomic classification is based on molecular biological methods of bacteria identification. In contrast, the previous classification, under which the genus Erwinia had been classified within the Family Enterobacteriaceae, was based on morphological and biochemical features of bacteria. It is widely acknowledged that molecular biological methods of bacteria identification are superior to methods based on morphological and biochemical features. This is equivalent to identifying a person by his/her genetic make-up as opposed to his/her external features and performance.

Organisms/microorganisms belonging to different taxonomic strata have unique behaviours and unique characteristics. These specific behaviours and features should be considered carefully when selecting appropriate methods for detecting microorganisms. Inappropriate selection of detection methodology leaves a very high possibility for a majority of microorganisms passing undetected, leading to erroneous conclusions about test samples.

If the fertiliser supplying company had the intention of clearing its good name that the shipment was free from Erwinia, the most appropriate methodology was to use specific, standard microbiological methods that are available to specifically detect the genus Erwinia or selected species of Erwinia. Or, the most logical and scientific approach should have been to test the sample for the presence of any culturable microorganism (using a standard microbiological technique suitable for this purpose) and if microorganisms were detected, to confirm their identity for genus Erwinia or species of Erwinia, subsequently. Instead what has been attempted through the test procedures that have been adopted by the SGS laboratory in Singapore was to try to convince the absence of Erwinia in an indirect way using a less sensitive and highly selective method targeting a particular group of bacteria with a questionable approach.

According to ISO 21528-1 (2017) and ISO 21528-2 (2017) test procedures, there is no specific guideline on the way of sampling. It is stated that the sampling is done on a case-specific way (if standard procedures are available for a given sampling material) and when such standards are not available, it is done via mutual agreement of the relevant parties.

According to the SGS report, the sample submitted for testing (Figure 2) is a parcel with approximate dimensions of 30 cm x 15 cm. The content within it could be estimated to be about 500 – 1000 g. It is highly questionable whether the amount of fertiliser sample submitted for testing was truly representative of the whole bulk of the 20,000 metric ton shipment. Furthermore, the number of samples submitted for testing seems to be only one, which is also not a sufficient number to represent the bulk of the shipment. There are widely-accepted sampling protocols that should have been used to obtain a series of representative samples. In scientific research, results based on a single sample are rarely, if ever, accepted as valid.

(See Figure 2: The sample used for testing as shown in the SGS test report)

ISO 21528-1 (2017) and ISO 21528-2 (2017) test procedures emphasise that the samples used for testing should be a proper representation of the bulk. In addition they provide clear guidelines on the quality of the sample at the time of its submission for testing. Accordingly, the sample should not have been damaged or changed during transport and storage. It is highly doubtful if the latter requirement could have been fulfilled from a shipment that had been stored in a bulk carrier for more than three months. Therefore, the validity/accuracy of the test results is questionable as the results do not reflect the initial quality/status of the shipment.

Based on above questionable/doubtful test procedures, it is not possible to conclude that the organic fertiliser sample tested by the SGS laboratory is free from plant, animal and human pathogens including Erwinia. The biggest concern arising from the conclusions of this nature is their negative impact on policy decisions and thereby the potential catastrophes that could be encountered by the nation as a whole. For example, the potential disease damage to a wide range of crops grown in Sri Lanka that could be caused by application of an organic fertiliser containing a plant pathogen such as Erwinia is enormous and could last for a very long period, incurring billions of rupees. Similarly, diseases to plants, animals and humans that could be caused by the wide range of microorganisms that are not detected by these highly-selective test protocols adopted by the SGS could lead to substantial economic and environmental damage along with human health hazards. These could take several years and several billions of rupees to be brought under control.

The meek response of the Sri Lankan authorities to the legal challenge of Qingdao based on the erroneous and questionable SGS test report from Singapore demonstrates their readiness to accept anything coming from an international agency while ignoring the scientifically-valid tests and conclusions of the Sri Lankan scientists. This sends a very negative detrimental signal to the national scientific community and to the nation as a whole.

References:

1. UK Standards for Microbiology Investigations: Identification of Enterobacteriaceae (2013), Issued by the Standards Unit, Microbiology Services, Public Health England, 32 pp.

2. Baruzzi, F., Cefola, M., Carito, A., Vanadia, S. and Calabrese, N., (2012). Changes in bacterial composition of zucchini flowers exposed to refrigeration temperatures. The Scientific World Journal, https://doi.org/10.1100/2012/127805, 6 pp.

3. Adeolu, M., Alnajar, S., Naushad, S. and Gupta, R.S., (2016). Genome-based phylogeny and taxonomy of the ‘Enterobacteriales’: proposal for Enterobacterales ord. nov. divided into the families Enterobacteriaceae, Erwiniaceae fam. nov., Pectobacteriaceae fam. nov., Yersiniaceae fam. nov., Hafniaceae fam. nov., Morganellaceae fam. nov., and Budviciaceae fam. nov. International Journal of Systematic and Evolutionary Microbiology, 66(12), 5575-5599.