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

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 Supplementary information The online version contains supplementary material available at 10.1007/s00253-022-12270-w.


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 (Galarde-López et al. 2022). These infectious diseases have an 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 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;Perez-Palacios 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 .
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 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 ChromoSelect Selective Agar Base, Simmons citrate agar (SCA) with 1% inositol, and HiCrome™ Klebsiella Selective Agar Base (van Kregten et al. 1984;Glupczynski et al. 2007;Charles et al. 2022). However, these media owing to their prohibitive cost, formulation complexity, and indistinguishable nature towards KpSC and other Klebsiella strains are not extensively used for routine surveillance of wastewater. Thus, the need of the hour is to develop a selective and differential medium that selectively grows and differentiates between the species belonging to the KpSC group and other genetically distant species of Klebsiella.
In this study, we sought to evaluate a novel medium termed Klebsiella blue agar in selectively promoting the growth of Klebsiella spp. and differentiating the members of the two cohorts. The components of the proposed medium are readily available and rationally put together to render it selective and differential. KBA was compared with KSA's ability to promote and differentiate the species. Also, compared to the KSA, the discriminatory power of the KBA medium in differentiating the Klebsiella sp. belonging to the group KpSC from other enteric bacteria for potential integration into routine surveillance workflow was studied using simulated sewage as the source of the environmental sample.

Media
The composition of Klebsiella blue agar media per litre of distilled water is as follows: solution A -3 g potassium dihydrogen phosphate (Sisco Research Laboratories Pvt. Ltd.), 6 g dipotassium phosphate (EMPLURA, Merck Life Science Pvt. Ltd.), 6 g sodium chloride (Sigma Aldrich), 64 mg methylene blue (Spectro Chem Pvt. Ltd.); solution B -100 mg magnesium sulphate dihydrate (MERCK Specialties Pvt. Ltd.) and 17 g agar (Hi Media Laboratories Pvt. Ltd.). Solutions A and B were separately autoclaved at 121 °C for 20 min. On cooling, filter sterilised bile salt, tryptophan, and glycerol 1.5 g/L, 2 g/L, and 0.2%, respectively, were added to solution A. KBA was constituted by mixing solutions A and B and poured onto sterile Petri dishes. The Luria Bertani broth (LB) (Hi Media Laboratories Pvt. Ltd.) and HiCrome™ Klebsiella Selective Agar Base (HiMedia Laboratories Pvt. Ltd.) were used for comparison.

Growth characteristics on Klebsiella blue agar
To assess the growth, respective cultures of Klebsiella spp. were streaked on the KBA media. The KBA plates were incubated at 37 °C for 24 h. The growth and morphology of the bacterial cultures in KBA after 48 h were also studied.
The tests were performed independently and in duplicate.

Selective and differential nature of KBA
All the available bacterial strains were grown overnight at 37 °C in LB broth and were used to check the ability of KBA to differentiate Klebsiella spp. from other enteric/non-enteric bacteria. The KBA media were compared to the commercial media -KSA, which is selective for Klebsiella species. Since all the selected bacterial cultures grow efficiently in LB, it was used as general media control. The cultures were streaked onto KBA, KSA, and LB agar plates and incubated for 24 h at 37 °C. Extended incubation of the plates was done for 48 h at 37 °C. The tests were performed independently and in duplicate.

Efficiency evaluation of KBA in surveillance of Klebsiella spp. in synthetic sewage
The efficiency of the KBA media in surveillance of sewage for the presence of Klebsiella spp. was checked in synthetic sewage augmented with bacteria as described in the literature (Salim et al. 2022) The augmented synthetic sewage was incubated for 1 h in a shaking incubator at 37 °C, following which the sample was plated at different dilutions onto the KBA, KSA, and LB media plates in triplicates and incubated at 37 °C for 24 h. Differences in the bacterial growth on all three media were analysed along with their respective CFU/ mL. One-step multiplex colony PCR of the isolated colonies using the primers specific for K. pneumoniae and K. quasipneumoniae (Table 1) was used to evaluate the differential and selective nature of all three media. Bacterial colonies with different morphologies were selected for the molecular analysis. Colonies were resuspended in 100 µL of sterile water and pre-treated at 90 °C for 30 min before adding primers and reagents. PCR amplification conditions were optimised and performed as described by researchers (Fonseca et al. 2017). Briefly, thermocycling conditions were as follows: 95 °C for 5 min, followed by 40 cycles at 94 °C for 30 s, 62.5 °C for

Statistical analysis
Statistical analysis was performed using the platform Graph Pad Prism 9.0. The data obtained were analysed using the one-way ANOVA test with Dunnett's multiple comparison tests to indicate any statistical significance between LB, KBA, and KSA. The significance level for the data sets was defined at p ≤ 0.05. All the data sets in the graphs are presented as mean values with respective standard deviations.

Media formulation
The components in KBA were judiciously included to render it selective and differential. Methylene blue, an inhibitor of the gram-positive organisms, was used to dissuade their growth in the medium. Bile salt and tryptophan were included as they are conducive to the growth of Klebsiella spp., leveraging on their ability to tolerate bile salt and metabolise tryptophan. Glycerol provided in the media acted as a sole carbon source suitable for Klebsiella spp. A total of 0.3% of NaCl rendered the media differential by imparting a dark green colouration to the Klebsiella spp. colonies (Table 2).

Colony morphology of Klebsiella spp. on the Klebsiella blue agar
Freshly prepared KBA medium appeared deep blue due to methylene blue. Selective and differential nature of the KBA agar K. pneumoniae, K. aerogenes, K. quasipneumoniae, K. variicola, and all MDR strains of K. pneumoniae showed a well-defined growth in the KBA medium (Fig. 5). All the colonies were green in colour, except for K. aerogenes, K. pneumoniae MDR K5, and K. pneumoniae U4677 which  Fig. 5m, o, n, r, p, and q] grew in the KSA with a light purple colour. However, we observed that P. vulgaris and A. baumannii [ Fig. 4p and q] showed the same purple pigmentation when cultured in KSA. E. coli ST155 [Fig. 3n] showed a scanty growth after incubation for 24 h. The colonies of S.

Selective isolation of Klebsiella spp. from synthetic sewage by KBA
The difference in the bacterial count of simulated synthetic sewage when plated onto the LB, KSA, and KBA media was determined. KSA had a lesser bacterial count of 3.87 × 10 7 CFU/mL than LB, with a colony count of 4.17 × 10 7 CFU/mL, while KBA being a more selective medium of the three had only 2.14 × 10 7 CFU/mL [ Fig. 6a]. Selectivity of the medium could be seen from the plates of LB, KSA, and KBA, where in apart from the decrease in the colonies in KBA, the colonies of the Klebsiella spp. are growing with their characteristic green colouration. The selective nature of the KBA was further confirmed by colony-based multiplex PCR using primers specific for K. pneumoniae and K. quasipneumoniae. The PCR result confirms that the colonies in the KBA media are of Klebsiella spp. [Fig. S1e, f, and g]. Even though the colonies growing in LB and KSA have Klebsiella spp., it also supports the growth of other bacteria present in the simulated sewage [ Fig. S1a, b, c, and d].

Increased selective nature of KBA over KSA
The replica plating technique was used to demonstrate the selective nature of the KBA medium over the KSA medium. LB medium had 253 CFU, of which only 209 CFU grew in the KSA medium when transferred [ Fig. 7a]. While in the KBA, out of 253 CFU, only 157 CFU grew when transferred [Fig. 7a]. The Venn diagram shows the relative growth comparison of the colonies in LB, KSA, and KBA [ Fig. 7b]. All the colonies in KBA medium were growing in KSA, but not all colonies found in KSA were growing in KBA but were present in the primary LB plate. Thus, it could be inferred that KBA is more selective than KSA, which was further confirmed using the one-step multiplex colony PCR. respectively; e, k, and q correspond to clinical strains of K. pneumoniae U4677, U4698, U4865, and OF9168 respectively; and f, l, and r correspond to K. variicola

Discussion
Sewage is a hotspot for antibiotic-resistant bacteria, and there is a need for routine monitoring of the sewage to assess the prevalence of bacterial pathogens, for example, Klebsiella spp. The genus is fast gaining attention for its resistance to last-resort treatment (carbapenem antibiotics) , which has led us to formulate a new differential and selective media for their isolation from environmental samples. The contribution of environmental reservoirs to the ever-increasing healthcare-associated Klebsiella infections and nosocomial outbreaks is increasing linearly (Perez-Palacios et al. 2021b). The effective monitoring and mitigative strategies demand discrimination of Klebsiella spp. falling into different cohorts. KpSC is a pathogenically important group that includes K. pneumoniae, K. quasipneumoniae, and K. variicola. The other Klebsiella spp., viz., K. aerogenes, K. oxytoca, K. indica, and K. terrigena, to name a few, constitute the other group.
The KSA medium has often been used to isolate Klebsiella spp. from diverse sources (Divakaran et al. 2019;Varshney et al. 2021). However, our preliminary study suggested that KSA supports the growth of P. vulgaris and A. baumannii in the same way as that of Klebsiella spp.
[ Fig. 4p and q], and it was difficult to distinguish them from each other. To tackle this problem, we ventured to develop a new selective medium that would support the growth of Klebsiella spp. while suppressing the growth of other gramnegative and most gram-positive bacteria. The new medium enabled the differentiation of the colonies of Klebsiella spp. belonging to the KpSC with a characteristic green colouration [ Fig. 5g-l].
The availability of different carbon sources supporting the growth of Klebsiella spp. was investigated in the study by van Kregten et al. (1984), Grimont and Grimont (2015), and Kumar and Park (2018), which was used as references for selecting the carbon source in the KBA medium. Both lactose and glycerol are known to support the growth of Klebsiella spp., hence were analysed as the sole carbon source in the KBA medium. It was observed that when compared to lactose, the selectivity improves with the inclusion of glycerol [Figs. 3,4,and 5]. On the other hand, lactose supported apart from Klebsiella spp., growth of P. vulgaris, E. coli ST155, A. baumanii, and S. enterica with the same characteristic green colouration as that of the Klebsiella colonies after 24 h of incubation. Glycerol is also known to support the growth of Clostridium pasteurianum, Clostridium butyricum, and K. aerogenes (Valan Arasu et al. 2011). However, methylene blue in the medium inhibited the growth of gram-positive bacteria like Clostridium spp. and Bacillus spp. [Fig. 4i]. The Klebsiella spp. metabolises the glycerol present in the medium, producing acid that precipitates the dye onto the growth surface, imparting a characteristic green sheen to the bacterial colonies. The KBA medium, being differential in nature, was able to differentiate between the growth of K. aerogenes from K. pneumoniae and K. quasipneumoniae, and K. variicola (KpSC complex). The colonies of K. aerogenes appeared as translucent mucoid colonies Fig. 6 Selective isolation of Klebsiella spp. on KBA from a heterogenous population in synthetic sewage. a Relative differences in the bacterial population on LB, KSA, and KBA were estimated and plotted (p ≤ 0.05) (Graph pad prism 9.0). The images b, c, and d correspond to the LB, KSA, and KBA media plates from which the colony count was estimated [Fig. 2a], which was different from the colonies of K. pneumoniae [ Fig. 1a], K. quasipneumoniae [ Fig. 5h], and K. variicola [ Fig. 5l]. It is pertinent to note that apart from Klebsiella spp., A. baumannii, P. vulgaris [ Fig. 4j and k], E. coli ST155, and P. fluorescens [ Fig. 3h and i] grew in the KBA medium scantily. Except for P. fluorescens, which grew with a blue colouration [ Fig. 2b], none of the other organisms showed any increased growth after 48 h. We observed during the formulation of the medium that an optimal concentration of NaCl improved the differential nature of the media. From a range of NaCl concentrations of 0.1 to 0.8%, 0.6% was found to be the optimal concentration.
A 0.15% concentration of bile salt is known to support the growth of gram-negative bacteria while inhibiting most of the gram-positive (Cremers et al. 2014), hence was used in the KBA medium to make it selective. The rationale for including tryptophan in the medium was to promote the growth of Klebsiella spp. amongst which many are indole positive, and the remaining can use tryptophan as a carbon or nitrogen source. The pH of the media is 7.20 ± 0.2, and the presence of K 2 HPO 4 and KH 2 PO 4 acts as a buffering component in the medium. MgSO 4 was added as a micronutrient which acts as a cofactor for enzymatic reactions. Moreover, all these features make the media non-conducive for competing organisms that exhibit no growth after 24 h of incubation. The composition of the chromogenic mixture in the KSA medium is proprietary and incurs high cost. KBA's composition is well defined without any complex mixture, containing only methylene blue, tryptophan, and bile salt as the selective and differential components. Hence owing to its readily available Relative differences in the bacterial population on KSA, and KBA, when replica plated from LB, were estimated, and plotted (p ≤ 0.05) (Graph pad prism 9.0). b Venn diagram indicating unique and shared colonies on LB, KSA, and KBA. c, d, and e correspond to the LB, KSA, and KBA media plates from which the colony count was estimated components, the medium can be easily formulated and rampantly used in laboratories.
KBA medium was able to selectively isolate Klebsiella spp. from simulated synthetic sewage augmented with bacteria (total bacterial count of 4.17 × 10 7 CFU/mL) of different genera, including nine strains of K. pneumoniae [ Fig. 6a and d]. Although KSA supported the growth of Klebsiella spp., it also favoured the growth of other bacteria (3.87 × 10 7 CFU/mL). It was confirmed using a one-step multiplex colony PCR with primers specific for K. pneumoniae and K. quasipneumoniae [ Fig. S1a to g]. The primers SHV-f and OKP-f are specific and target the chromosomal class A β-lactamase gene bla SHV and bla-OKP of K. pneumoniae and K. quasipneumoniae [ Table 1] respectively. The reverse primer DeoR-r is derived from a gene coding for an ATPase that is part of the stable bacterial genome and flanks the respective bla SHV and bla OKP genes (Fonseca et al. 2017). One-step multiplex colony PCR showed that KBA is relatively more selective than KSA.
Replica plating of LB plate onto KSA and KBA showed that KBA was more selective for Klebsiella spp. than KSA medium. Out of the 253 colonies [ Fig. 7a and b], only 157 colonies grew on the KBA medium, while 209 colonies grew on the KSA medium, out of which 52 colonies were unique to KSA but not to KBA. These results indicated the selective nature of the medium.
KBA owing to its proven characteristics, namely facile formulation, improved selectivity, differential nature, and cost-effectiveness, could enhance its potential to be used in environmental settings such as wastewater. Furthermore, elaborate field testing and exploration of its commercial viability can lead to efficient translation for monitoring, surveillance, and basic research applications.