A novel and improved selective media for the isolation and enumeration of Klebsiella species

Applied Microbiology and Biotechnology https://doi.org/10.1007/s00253-022-12270-w METHODS AND PROTOCOLS A novel and improved selective media for the isolation and enumeration of Klebsiella species Megha Prasad1 · Sindhu K. Shetty1 · Bipin G. Nair1 · Sanjay Pal1 · Ajith Madhavan1 Received: 1 September 2022 / Revised: 28 October 2022 / Accepted: 31 October 2022 © The Author(s) 2022 Abstract Bacterial pathogens are fostered in and transmitted through wastewater. Hence, monitoring their impact on sanitation and hygiene is imperative. As part of the monitoring process, culture-based methodologies are primarily used, which centre on the use of selective and differential media. Media available today are, at best, difficult to formulate and, at worst, prohibitively expensive. To address this lacuna, the study proposes a selective and differential medium for Klebsiella spp. Klebsiella blue agar (KBA) is completely selective against selected gram-positive bacteria (Bacillus spp., Staphylococcus aureus) and a few gram-negative bacteria (Acinetobacter baumanii, Serratia marcescens). On the other hand, it supports the growth of the chosen members of the Klebsiella pneumoniae species-complex with a characteristic green colouration. Methylene blue, tryptophan, and bile salt make up the selective components of KBA. Moreover, methylene blue, 0.6% NaCl, and glycerol render it differential. KBA was more selective than HiCrome™ Klebsiella Selective Agar Base (KSA) in replica plating experiments. KBA promoted only 157 CFUs against 209 CFUs in KSA when stamped with 253 CFUs grown on LB. The colonies so isolated were predominantly Klebsiella spp., on identification through colony polymerase chain reaction. Moreover, the differential nature of KBA distinguished Klebsiella aerogenes from other species. On the contrary, KSA lodged colonies indistinguishable from each other and Klebsiella spp. Due to its ease of formulation, high selectivity, differential nature, and cost-effective composition, KBA is a viable option for the routine culture of Klebsiella spp. in environmental and clinical settings. Key points • Formulated a novel selective and differential media for Klebsiella spp., named Klebsiella Blue agar • Facile formulation methodology • Can be employed to isolate Klebsiella spp. from complex sources such as wastewater Keywords Wastewater surveillance · Selective medium · Klebsiella spp. · Differential medium Introduction Pathogens of public health importance transmitted through direct or indirect contact between humans, animals, and the environment are the leading cause of emerging and re-emerging infectious diseases all over the globe (GalardeLópez et al. 2022). These infectious diseases have an * Sanjay Pal * Ajith Madhavan 1 School of Biotechnology, Amrita Vishwa Vidyapeetham, Kerala 690525, India adverse impact on global economies and public health (Jones et al. 2008). Amongst these infectious agents, the Enterobacteriaceae family has been fast gaining attention as this group has been linked to a high percentage of hospital-acquired infections, and most antibiotics are often ineffective against them (Babu et al. 2016; Sakkas et al. 2019; Rolbiecki et al. 2021). Klebsiella genus, a class of gram-negative, encapsulated, non-motile bacteria belonging to the Enterobacteriaceae family (Dworkin et al. 2006; Grimont and Grimont 2015; Wyres et al. 2020) is one of the leading causes of nosocomial and community-acquired infections. Klebsiella spp. is grouped into cohorts, namely Klebsiella pneumoniae species complex (KpSC), which includes Klebsiella pneumoniae, Klebsiella quasipneumoniae, and Klebsiella variicola, while 13 Vol.:(0123456789) Applied Microbiology and Biotechnology Klebsiella oxytoca, Klebsiella indica, and Klebsiella terrigena (Dong et al. 2022) into another genetically distinct group. The KpSC group of bacteria is responsible for most nosocomial and community-acquired pneumonia, urinary tract, and bloodstream infection associated with Klebsiella spp. in healthcare-associated settings (Prado et al. 2008; Stojowska-Swędrzyńska and Krawczyk 2016; Martin and Bachman 2018; Dong et al. 2022). These bacteria can thrive in various niches, including plants, animals, and waterbodies (Holt et al. 2015). They have an uncanny ability to exchange their plasmid with other species. This property and high genomic plasticity make these species a reservoir of virulence and antimicrobial resistance genes (Ramirez et al. 2014). The World Health Organisation in 2017 declared the extended-spectrum β-lactam (ESBL)producing and carbapenemase-producing Klebsiella spp. a latent threat to public health due to its ability to accumulate multidrug resistance (MDR) and hypervirulence (Zhou et al. 2016), especially in wastewater which is a hotbed for acquiring and disseminating MDR genes (Moges et al. 2014; Gomi et al. 2018; Bonardi and Pitino 2019; PerezPalacios et al. 2021a). Nutrient-rich wastewater and waterbodies where the bacterial cell density is exceptionally high, the factors influencing the increase in antibiotic resistance in bacteria are enhanced; for example, hospital effluents are an ideal pool for exchanging resistance genes between clinical and environmental bacteria (Sakkas et al. 2019). Outside of the clinical settings, little is known about the ecology and transmission of Klebsiella spp.; hence, the detection, identification, and monitoring of Klebsiella spp. and their different clonal groups in the environment and effect on humans remain undefined (Mathers et al. 2015; Holt et al. 2015). Understanding the emergence and spread of these antibiotic-resistant bacterial strains in the environment requires wastewaterbased epidemiological monitoring and surveillance system (Hornsey et al. 2013; Daughton, 2020; Galarde-López et al. 2022). Molecular DNA-based techniques like pulsed-field gel electrophoresis, multilocus sequence typing, repetitive element sequence-based PCR, and whole genome sequencing (Dinkelacker et al. 2018) are currently employed for this purpose. However, being laborious and cost-limiting, these high-end techniques are restricted to research rather than routine real-time surveillance (Rossen et al. 2018). Developing a cost-effective, easy-to-formulate, selective, and differential bacterial culture media is imperative to make wastewater monitoring and surveillance more rigorous and hassle-free. A primary medium is rendered selective and differential by adding components such as dyes, chemicals, and antibiotics. However, rising antibiotic resistance and lack of exploration of new chemical additives as selective agents have curtailed the development of a new and improved selective medium. Over the years, many different selective culture 13 methods have been proposed for active surveillance of K. pneumoniae and its associated clonal groups in different settings. These include but are not restricted to — MacConkey agar supplemented with ceftazidime, Klebsiella Chr (...truncated)