Phenotypic Changes in Phage Survivors of Multidrug-Resistant Klebsiella pneumoniae

Abstract

Multidrug-resistant Klebsiella pneumoniae (MDR-KP) infections have become a major global issue in the healthcare sector. Alternative viable tactics for combating bacterial infections, such as the use of bacteriophages, can be considered. One of the major challenges in phage therapy is the emergence of phage-resistant bacteria. This study isolated bacteriophages from water and soil samples against MDR-KP isolates. Susceptible bacterial hosts were exposed to phages at different concentrations and prolonged durations of time to obtain phage-resistant survivors. Phenotypic changes such as changes in growth rates, biofilm formation ability, antibiotic sensitivity patterns, and outer membrane proteins (OMPs) profiling of the survivors were studied. Our findings indicate that the phage ØKp11 and ØKp26 survivors had reduced growth rates and biofilm formation ability, altered antibiotic sensitivity patterns, and reduced OMPs expression compared with the parent MDR-KP002 isolate. These results suggest that the alternations in the bacterial envelope result in phenotypic phage resistance among MDR bacterial isolates.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12088-024-01217-6.

Multidrug-resistant Klebsiella pneumoniae (MDR-KP) causes a range of high-mortality illnesses, including pneumonia, urinary tract -infections, and bloodstream infections. Almost 80% of nosocomial infections caused by these strains are resistant to antibiotics, and the mortality rate due to their infection is as high as 50% [1]. For survival and immune evasion, K. pneumoniae uses a range of virulence factors including but not exclusive of capsule polysaccharide, lipopolysaccharide, fimbriae, outer membrane proteins (OMPs), determinants for iron acquisition, nitrogen source utilization and thick biofilms [2, 3]. K. pneumoniae produces two major OMPs, namely OmpK35 and OmpK36, which play significant roles in the permeability of antimicrobial agents and substrates, and in interactions with the host defence mechanisms [4]. Phage therapy has resurfaced as a viable alternative for treating recalcitrant K. pneumoniae infections, especially after the advent of MDR-KP [5]. Several phages use the outer membrane proteins as receptors [6–8]. Modifying OMPs in Gram-negative bacteria may lead to removing or changing the phage receptor, preventing phage adsorption and inhibiting infection [9]. Phages may also evolve to adapt to changes in the OMPs receptor site [10]. In this study, the phenotypic characteristics of phage-resistant survivors (PRS) that developed in MDR-KP clinical isolates following exposure to isolated phages were studied.

Viable count reduction was assayed at 0.01 multiplicity of infection (MOI) for lytic phages ØKp11 and ØKp26 on host KP002 (an anonymized MDR-KP clinical isolate). The mid-exponential growth cultures incubated with shaking (120 rpm) at 37 °C were infected with phages. Hourly OD₆₀₀ measurements and colony enumeration were conducted for testing and control. Colonies that appeared on plates after an OD₆₀₀ drop were screened for PRSs. PRS-ØKp11 and PRS-ØKp26 were sub-cultured consecutively ten times on nutrient agar, and the culture was tested for phage sensitivity by the spot assay. Cultures resistant to the phages that the parent strain was susceptible to were considered as PRS and taken for further evaluation.

Growth patterns of the PRSs were compared with the parent cultures by recording the OD₆₀₀ values every 20 min up to 6 h. The biofilm formation ability was determined and interpreted as per established procedures [11]. All experiments were performed in triplicate and repeated for three biological replicates. The Minimum inhibitory concentration (MIC) was determined for PRS-ØKp11, PRS-ØKp26, and parent KP002 isolate using broth micro-dilution method for the antibiotic ceftazidime (Sigma-Aldrich, USA) as per the Clinical & Laboratory Standards Institute (CLSI) guidelines [12]. Mitomycin C at 1 µg/ml was used to induce prophages in the parent culture as well as the PRS. Spot assay of PRS culture lysates on the KP002 was performed to determine if the PRS were lysogens of the phages. The OMPs were extracted from KP002 and PRS-ØKp11 and PRS-ØKp26 using sarkosyl L (N-lauroylsarcosine) (Sigma-Aldrich) [13]. The concentration of extracted protein was measured using a nano spectrophotometer (IMPLEN, USA), and 20 µg of proteins was separated by 12% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Mid-range protein ladder (Genei, India) at 13 µg served as the marker and loading control for the gel. The protein bands were visualized using Coomassie brilliant blue R-250 (HiMedia, India) staining and documented (Uvitec, UK -MJEA13553).

KP002 growth was a typical sigmoid curve. When infected with phage, OD₆₀₀ and CFU/ml were reduced approximately 2 h after phage infection (Fig. 1). Phage-resistant bacterial colonies emerged for isolate KP002 approximately 18 h post phage infection with ØKp11 and ØKp26. PRS—ØKp11 was resistant to lysis by ØKp11 and other phages (phage ØKp1, ØKp6, ØKp10, ØKp12, and ØKp26), which were active on the parent KP002. However, both parent and resistor isolates were sensitive to ØKp25 (Fig. 2a and b). PRS–ØKp26 was resistant to ØKp26 and all other phages as tested by spot assay (Fig. 2c and d).

Fig. 1.

Viable cell reduction assay. CFU/ml of infected and non-infected bacterial culture with bacteriophages at 0.01 MOIs is compared

Fig. 2.

Comparison of phage survivors. Spot assay of different phages a KP002 Control b PRS-ØKp11 c KP002 Control d PRS-ØKp26. e Growth curves for control KP002 and its two phage survivors. f Biofilm formation ability of KP002 and its two phage survivors. g OMP profile of control KP002 and its two phage survivors. M—mid range protein marker (Genei), L1—KP002 control, L2—PRS-ØKp11, L3—PRS-ØKp26

The growth rate of the parent culture was significantly higher than the PRSs (PRS-ØKp11 and PRS-ØKp26) as determined by the two-tailed test (P value 0.0006 and 0.0003 respectively), and the strength of biofilms formed was reduced in case of PRS isolates (Fig. 2e and f). The MIC of ceftazidime was reduced for both PRS compared to the parent KP002 (Supplementary file -Table 1). Isolate KP002 and its two PRS were positive for the presence of bla_SHV and bla_TEM genes, and hence the increased susceptibility was not due to the loss of resistance genes. Mitomycin C culture lysates of the PRSs did not show lytic activity on the parent culture KP002 indicating the absence of lysogen-mediated immunity. The SDS-PAGE profile for total OMPs showed several bands between 29 and 66 kDa (Fig. 2g), two bands can be compared to OmpA (~ 30 kDa) and the major porins OmpK35/OmpK36 (~ 35 kDa) in K. pneumoniae as reported by several authors [4, 14–18]. The intensity of the band corresponding to the one suspected to be major OmpK35/36 was reduced in PRS isolates as compared to the KP002 control isolate while major variations were not observed in either the number of bands or intensity in other OMPs when compared to the control.

Understanding the phage-host interaction and population dynamics is crucial for successful phage therapy development and application. It is important to consider the evolution of phage-resistant bacteria [19, 20]. Changes in bacteriophage sensitivity patterns among the PRSs can be attributed to the decrease in the phage adsorption into bacterial cells, which may be affected by changes in bacteriophage-binding receptors such as lipopolysaccharides (LPSs), OMPs, capsules, and bacterial appendages [21, 22].

Mutations in genes whose products are involved in the synthesis/assembly of cell surface structures can lead to loss of phage receptors leading to impaired phage adsorption [23]. For example, deficiency of capsular polysaccharides in hypervirulent K. pneumoniae renders phage resistant strains with slower growth dynamics and increased biofilm production compared with the parent strain [24]. In the case of phage-resistant mutants of Pseudomonas aeruginosa, T4P assembly deficient strains showed lower biofilm production while LPS O-antigen biosynthesis deficient ones showed a threefold increase in biofilm formation than wild type [25].Our findings support an earlier study on phage-resistant P. aeruginosa PAO1 isolates, which demonstrated lower biofilm production and impaired bacterial growth compared to the wild type. This is likely due to a trade-off that occurs in the emergence of phage resistance that hampers their ability to proliferate and form biofilms [26].

The bacterial LPS layer and OMPs act as primary and secondary phage receptors in Shigella flexneri, and strains lacking both OmpA and OmpC showed decreased plating efficiency and slower rates of phage infection [6, 10]. The changes in bacteriophage receptors can influence antibiotic resistance phenotypes in a pleiotropic way, and the advent of bacteriophage-insensitive bacteria can enhance antibiotic resistance [21, 24]. In this study, MIC values for both PRS isolates for antibiotic ceftazidime were reduced even though the bacteria retained antibiotic resistance. A similar observation for phage-resistant bacteria is reported in colistin-resistant Acinetobacter baumannii. The phage-resistant isolates were less virulent compared to the parental strains [27].

In K. pneumoniae, loss of OmpK36 alone results in enhanced MICs to cephalosporins and decreased virulence, whereas loss of both OmpK35 and OmpK36 increases resistance to cephalosporins, loss of fitness and virulence with impaired growth supported by animal studies [28, 29]. However, in the present study, the SDS-PAGE banding patterns of total OMPs showed no major difference among the parent and PRS isolates except the band around 36 kDa. The sarkosyl method of extraction provides total OMPs and cannot separate individual OMPs in the mixture. Further confirmation with LC MS/MS or Western blotting is required to identify the altered expression of specific OMPs 35/36. Our data further support previously reported findings that OmpK36 is one of the major receptors needed for Klebsiella phage infection [30] and adsorption [18]. Reporting with a single strain of K. pneumoniae limits our findings. PRSs of K. pneumoniae show altered expression of OMPs, decreased MIC for ceftazidime, and reduced capacity to form the biofilm as compared with the parent isolate. A detailed exploration to understand the evolution of PRSs and studies into mechanisms that reverse or advert resistance to the phage would empower phage therapy.

Supplementary Information

Below is the link to the electronic supplementary material.

Declarations

Conflict of interest

The authors declare that they have no conflicting interests associated with this publication.