Sugar-fermenting flora mask Vibrio cholerae in Kligler iron agar

Cholera is a severe diarrheal disease caused by Vibrio (V.) cholerae, a Gram-negative, motile bacterium. The pathogen colonizes the small intestine and produces an exotoxin that disrupts sodium transport, leading to massive fluid loss, dehydration, and potentially death if untreated[1,2]. Kligler iron agar is used to differentiate enteric bacteria and can serve as a short-term transport medium for suspected V. cholerae. It contains glucose, lactose, and phenol red as a pH indicator. Glucose fermentation turns the butt yellow (anaerobic), while lactose fermentation turns the whole medium yellow due to sustained acid production[3]. In the current study, V. cholera O1 serotype ogawa were isolated from eight Iraqi patients in Al-Shomali General Hospital, Babylon city, Iraq. Isolates were obtained from watery diarrheal stool and then cultured in peptone water for 8 h, followed by overnight incubation on blood agar, MacConkey agar and TCBS agar (HI Media, India) to differentiate between different species. They were biochemically tested using oxidase, indole, urease, simmons citrate, Kligler iron agar, Gram staining, string test and antisera polyvalent ogawa and inaba (Hardy Diagnostics, USA). Isolates were confirmed by VITIK-2[4]. Eight from all these microbes including Escherichia (E.) coli, Klebsiella spp., Enterococcus spp., Staphylococcus aureus, Pseudomanas spp., Bacillius spp. and Candida spp. and V. cholera isolates were obtained from watery diarrheal stool then cultured in peptone water for 8 h. V. cholerae was cultured together with an equal quantity of each bacterial species in Kligler iron agar for 24 h by taking a single colony of V. cholerae and a single colony of the other bacteria. The cultures were then incubated on blood agar for 24 h to observe the growth of V. cholerae compared to the other bacteria by direct colony counting for growth rate comparison. For statistical analysis, SPSS software 26 (SPSS Inc., Chicago, USA) was used. Chi-square test was used to represent the data with P value<0.05 considered significant. The results of this study showed the growth rate comparison between total growth rate of all bacterial isolates (75%) and V. cholera (25%) with a significant difference (P<0.0001) indicating that other bacteria inhibit the growth of V. cholera while the proposed rate of growth should be 50% for V. cholera and each other bacterium. A growth rate comparison between V. cholerae and various microbial isolates revealed significantly lower growth of V. cholerae on blood agar after co-culture in Kligler iron agar. The relative growth percentages of V. cholerae compared to other species were as follows: 0% V. cholerae vs. 100% Candida spp. (P<0.000 1), 1.5% V. cholerae vs. 98.5% Klebsiella spp. (P<0.000 1), 4.0% V. cholerae vs. 96.0% E. coli (P<0.000 1), 30.0% V. cholerae vs. 70.0% Bacillus spp. (P<0.000 1), 57.5% Enterococcus spp. vs. 42.5% V. cholerae (P=0.004 0), and 52.5% Staphylococcus aureus vs. 47.5% V. cholera (P=0.0400). No significant association between V. cholera (50.0%) and Pseudomonas spp. (50.0%) was found (P=1.000 0, Table 1).Table 1: Growth rate comparison between each bacterial isolates and Vibrio cholera.As we know V. cholera prefers an alkaline environment and is inhibited in acidic environment. This study concluded that when V. cholera mixed with bacteria capable of lowering the pH to a high acidic level in Kligler iron agar, which leads to the killing or slowed growth of V. cholera, depending on these ability of bacteria to acidify the environment. Another study also discussed the effects of acid shock on V. cholera that mentioned the exposure of V. cholera to mild acid shock (pH 5.5) slightly reduced cell viability[5]. Another study also revealed when bacteria were subjected to low acidic pH of the gastric environment, they might enter a viable but non- culturable state for survival[6]. In the current study, bacteria that ferment both glucose and lactose produced low pH that leads to strong inhibition of V. cholera growth as observed with E. coli and Klebsiella. Bacteria that ferment only glucose moderately inhibit V. cholerae growth. In contrast, bacteria that neither ferment glucose nor lactose, such as Pseudomonas aeruginosa, have no effect on V. cholerae growth. Candida albicans tends to ferment sugar rapidly, fully inhibiting the growth of V. cholerae. In conclusion, bacteria that ferment sugars or utilize sugars and turn pH acidic in Kligler iron agar can kill or inhibit V. cholera. Therefore, when V. cholera contaminated with another fermentable normal flora in Kligler iron agar that sent by terminal laboratories for confirming the result in central laboratory, V. cholera may be killed or inhibited by high acidity due to sugar fermentation in Kligler iron agar. Conflict of interest statement The author claim there is no conflict of interest. Authors’ contributions Alhusayni AA conceptualized and developed the study design, reviewed the literatures, took part in proposal development, led the data collection and data entry, and took part in data analysis and interpretation, manuscript writing and review. Al-Khikani FHO also conceptualized and developed the study design, participated in data analysis and interpretation, engaged in proposal development, and contributed to manuscript preparation and review. All authors have read and approved the final version of the manuscript. 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