Isolation and polymerase chain reaction identification of bacteria from the 2014–2015 flood of Kota Bharu, Kelantan, Malaysia

Charts, Tables, and Figures can be requested by email from: pwaveno.hb@umk.edu.my; pwaveno.bamaiyi@kiu.ac.ug. PCR results on gel electrophoresis are shown ( Figures 3-7). The chart (Figure 8)classified the samples into 3 bacterial domains, Proteobacteria (67%), Firmicutes (32%), and Bacteroidetes (1%). The chart reveals 12 families of bacteria: Moraxellaceae (10%), Aeromonadaceae (8%), Comamonadaceae (13%), Neisseriaceae (2%), Bacillaceae 1 (16%), Staphylococcaceae (8%), Bacillales Incertae Sedis XII (3%), Bacillaceae 2 (3%), Streptococcaceae (2%), Flavobacteriaceae (2%), Enterobacteriaceae (25%) and Pseudomonadaceae (10%) with P = 0.03 for the number of isolates belonging to the families. Please see: https://16s.geneious.com/16s/results/ cdee2e80-b6b7-4a5c-bf33-4e6760294758.html for a version that can be manupulated. A literature search indicates that 10 of the 12 families of bacteria (83%) contained bacteria pathogenic to man and animals. The isolate sequences that passed the criteria, were successfully submitted to the GenBank, their accession numbers and species identified are listed in Table 1. There was no significant difference (P > 0.05) between the number of different species of bacteria isolated from the 6 locations studied. The susceptibility of 17 selected bacteria isolates to 7 different antibiotics was found to be significant with P < 0.0001 (Tables 2 and 3) with the bacteria having the highest susceptibility to gentamycin followed by tetracycline, and the lowest susceptibility was to penicillin G followed by ampicillin (Figure 9). The size of this difference was large (η² = 0.236). Some bacterial isolates in Table 2 (isolates 4d2 and 5d1; 4e2 and 6a1y; 6b2 and 6c) did not show the same antibiotic sensitivity pattern even though they were same bacterial species. Klebsiella pneumoniae showed the highest resistance to multiple antibiotics while Exiguobacterium mexicanum, Acinetobacter calcoaceticus, and Pseudomonas vranovensis did not show resistance to any of the antibiotics tested; 82% of bacteria isolated showed resistance to one or more antibiotics while 76% of bacteria isolates tested showed multiple antibiotic resistance. Readers can use the LINK and click on the different classes, families and/or species to find out more details about the abundance of each category among the samples. This will provide a comprehensive illustration of the samples.

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The unexpected nature of this study, which was conducted during the course of a devastating flood, may have affected some parameters that could have improved this study. The use of only culturing of water samples and looking for only bacterial colonies that grew within 48 hours may have excluded other bacteria such as Leptospira spp, Vibrio spp, and Burkholderiapseudomallei from being documented. Nevertheless, the isolation of several species of bacteria and genera of bacteria indicates a rich bacterial biodiversity of pathogenic or potentially pathogenic bacteria in the flood water during the great flood of December 2014 to January 2015 in Kelantan, Malaysia.

After the flooding, the prevalence of bacterial infections usually increases triggering episodes of intestinal symptoms such as diarrhea, vomiting, stomach aches, and other gastrointestinal disease symptoms as contaminated water moves from one geographical location to another, carrying a “cocktail” of bacteria along with it [16-18]. The distribution of the bacteria isolated did not show any remarkable difference between the different locations from which water samples were taken. This is probably because there was no large difference in the elevation between the locations, and during flood there is a massive movement of water from one location to the other, which can carry and distribute bacteria almost uniformly from one flood water location to the other. Different water depths would be expected to produce a variety of bacteria species [19]. This has potential public health implications because it implies waterborne infections can easily be carried from one flood location to another.

The predominance of proteobacteria making up 67% of bacteria in this study is similar to a study conducted in Thailand, which reported that majority of bacteria from the 2011 Thailand flood were from the phylum proteobacteria, which made up 56.5% to 91.4% of bacteria in different water samples in Thailand [6]. However, the majority of families and genera of bacteria reported from Thailand differ from the ones in this study, demonstrating the heterogeneity of bacterial communities across different flooded environments. Enterobacteriaceae from the phylum proteobacteria were the most common bacteria encountered in water samples from this study. Enterobacteriaceae also constitute the majority of waterborne and foodborne infections known in man and animals. Their ubiquitous nature and ability to thrive of the environment, helps them to be widespread in nature. They have been reported in various kinds of water, including flood water, by other investigators worldwide [16, 17, 20]. They are usually opportunistic bacteria, but can enter animals and human hosts, where some cause illnesses such as salmonellosis, shigellosis, and other intestinal infections. The high prevalence of this family in our study is a cause for concern because several members are known to be resistant to many antibiotics including those considered the current last line of antibiotic defense [21]. The present study has revealed Klebsiella pneumoniae subsp. Rhinoscleromatis as most resistant to the antibiotics tested. This is consistent with findings worldwide [22-24]. Three bacteria, Exiguobacterium mexicanum, Acinetobacter calcoaceticus, and Pseudomonas vranovensis did not show resistance to any of the antibiotics tested. This is consistent with other findings, but little is known about the antibiotic sensitivities of the relatively new bacterial species Pseudomonas vranovensis [25-27]. These bacteria species seem not to pose threats to health as do Klebsiella pneumoniae.

Flood water is a conglomeration from different sources including overflowing seas, rivers, streams, springs, wells, and other water. Humankind′s activities during floods including bathing, swimming, washing, and excretion of waste into flood water affects its bacterial composition [28]. Some of the pollution may also have come from industrial waste, sewage water contamination, and admixtures of water from all manner of unhealthy sources during the course of the flooding [29, 30]. The study from Thailand showed pathogenic bacteria and high cross-contamination between flood water and other water sources [5]. The degree of pollution of soil surface and the metallic components of the soil also determine the richness and diversity of the bacteria present with presence of zinc decreasing both diversity and levels of species richness [31]. The rich bacterial diversity of our flood water showed bacteria from human and animal sources, and bacteria from the environment. A study in Brazil found that usual environmental water is a rich source of many species of bacteria with varying degrees of antibiotic resistance, showing some bacterial communities tolerating up to 600 times the clinical treatment levels of common antibiotics [32].

There appeared to be some strain variation in pathogenicity of some of the isolates as indicated by the same species showing different antibiotic sensitivities. Further studies that go beyond species identification to identification of different strains and genes coding for resistance are required to establish if indeed these are different strains of the same bacteria with varying pathogenicity.

There were 9 bacteria in the present study not previously reported from any source in Malaysia based on our literature search. All of them were bacteria recently reported and classified within the last 20 years. Wautersia numazuensis (Cupriavidus numazuensis) was first reported in 2011 from Mexico in soils and agricultural plants [25, 33-37]. The epidemiological and public health impact of these water microbes and their ecological roles require future study.

During this massive flood session, there was a rich bacterial biodiversity including some species of potentially pathogenic bacteria that could endanger public health. This altered bacterial composition of normal water outside of flooding, and may explain why there are outbreaks of various infectious diseases during and after flood disasters. During the flood disaster period the only functional tertiary hospital (Hospital Universiti Sains Malaysia) in Kelantan handled 180 cases/day in the emergency department [38]. In adjacent Pahang state, 1,220 flood-related cases were handled within the first 6 days of the Kelantan flood disaster [39].

Most of the bacteria isolated from this study were resistant to one or more commonly used antibiotics. This is of interest to health practitioners and health policy makers because the presence of multidrug resistant bacteria should guide clinicians in the choice of antibiotics during flood disasters for effective treatment and control of waterborne infections. Gentamycin and tetracycline antibiotic classes appeared to be the best antibiotics to consider, but this may be an ever-changing picture. During flooding human and animal contact with flood water should be minimized, if not avoided completely, and adequate provisions should be made for provision of clean water to avoid outbreaks of waterborne diseases.