Susceptibility of colistin-resistant pathogens to predatory bacteria

Abstract

The increase in multidrug-resistant Gram-negative bacterial infections has forced the reintroduction of antibiotics such as colistin. However, the spread of the plasmid-borne mcr-1 colistin resistance gene have moved us closer to an era of untreatable Gram-negative infections. To evaluate whether predatory bacteria could be used as a potential therapeutic to treat this upcoming threat, the ability of Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus to prey on several clinically relevant mcr-1-positive, colistin-resistant isolates was evaluated. No change in the ability of the predators to prey on free swimming and biofilms of prey cells harboring mcr-1 was measured, as compared to their mcr-1 negative strain.

1. Introduction

Countless lives have been saved in the last 70 years since the introduction and use of antibiotics in medicine. However, in the last several years a troubling increase in infections caused by extensive drug-resistant (XDR) bacteria has been reported. It is estimated that more than 23,000 people die in the United States annually as a result of an XDR infection [1]. The rise in XDR infection caused by Gram-negative pathogens such as Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa has brought about the reintroduction of antibiotics that were previously discontinued because of their toxicity, one of which is colistin [2]. Colistin (polymyxin E) is a polycationic peptide that targets the bacterial electronegative phosphate groups associated with the lipid A part of the lipopolysaccharide (LPS). In 2015, the first plasmid-borne colistin resistance gene mcr-1 was reported in China [3], and is now believed to have worldwide distribution. With the growing risk of mcr-1 spread and the rise of untreatable infections, the need to develop new approaches to control infection is essential. One such approach which has gained attention in the last few years is the use of Gram-negative predatory bacteria from the genera Bdellovibrio spp. and Micavibrio spp. Bdellovibrio bacteriovorus and Micavibrio aeruginosavorus are motile Delta- and Alpha-proteobacteria, respectively, which prey on other Gram-negative bacteria [4]. In the last few years, several reports have been published that highlight the ability of predatory bacteria to control clinically relevant pathogens [5,6] and biofilms [7,8]. The nontoxic attributes of the predators were demonstrated on human cell lines, in ex vivo wound healing models and in several animal models of infection [9-13]. The ability of predatory bacteria to reduce K. pneumoniae numbers in the lungs of rats as well as Shigella flexneri in zebrafish has also been reported [12,14].

It is believed that, in order to feed, predatory bacteria first need to attach to the outer membrane of their prey; however, the specific mechanisms that govern prey specificity are not well known. Although the ability of predatory bacteria to prey on defined clinical isolates of XDR bacteria was reported [6,15,16], the ability of predatory bacteria to prey on mcr-1-positive, colistin-resistant isolates has not yet been investigated. Importantly, as mcr-1-mediated colistin resistance is mediated by LPS modification [17,18], it is possible that this surface chemistry alteration may prevent the predators to attach to the prey. In this study, the ability of predatory bacteria to prey on several mcr-1-bearing, LPS modified, colistin-resistant Gram-negative pathogens and their non-transgenic counterparts was assessed to determine the potential to use predatory bacteria to control this emerging threat.

2. Materials and methods

2.1. Bacterial strains and growth conditions

The following bacteria were used: A. baumannii ATCC 17978, Escherichia coli ATCC 25922, K. pneumoniae ATCC 13883 and P. aeruginosa ATCC 47085. In addition, we have evaluated each of the above recipient bacteria harboring a colistin-resistant gene mcr-1 which was placed on the recombinant plasmids pMQ124-mcr-1, or pMQ124XLAB1-mcr-1 for A. baumannii. The mcr-1 plasmid-bearing strains were previously verified as colistin-resistant and had LPS modifications [17]. Bacteria were grown at 37 °C in lysogeny broth (LB). Bacteria harboring the mcr-1 plasmid were cultured on LB supplemented with 50 μg/ml gentamicin. Colistin susceptibility of the isolates was confirmed by plating the bacteria on LB agar plates supplemented with 10 μg/ml of colistin. The predatory bacteria used were B. bacteriovorus strains HD100 (ATCC 15356), 109J (ATCC 43826) and M. aeruginosavorus strain ARL-13 [19]. Predatory bacteria were cultured and maintained as previously described [5,6,15], using E. coli strain WM3064 as prey. Predator stock-lysates were made by co-culturing the predators with prey cells in HEPES buffer (25 mM) supplemented with 2 mM CaCl₂ and 3 mM MgCl₂. Co-cultures were incubated at 30 °C for 24 or 72 h for B. bacteriovorus and M. aeruginosavorus respectively, until the culture cleared (stock-lysates). Stock-lysates were filtered through a 0.45-μm Millex pore-size filter (Millipore) to remove any remaining prey and used in predation experiments (harvested predators).

2.2. Predation experiments

To compare the susceptibility of colistin-resistant and susceptible isolates to predation, co-cultures were prepared by adding 0.4 ml of HEPES washed prey cells (~1 × 10⁸ CFU/ml) to 0.4 ml of harvested predators (~1 × 10⁸ PFU/ml for B. bacteriovorus and ~1 × 10⁷ PFU/ml for M. aeruginosavorus) and 3.2 ml HEPES medium. Predation was measured by the change in prey population enumerated by dilution plating during a 48 h incubation period [6,15]. Experiments were conducted twice in triplicate. Graphpad Prism 6 software was used to perform Student's t-test statistical analysis. P < 0.05 was considered statistically significant.

2.3. Biofilm predation and removal

Predation on surface-attached prey was conducted as previously reported [7,8]. A. baumannii and E. coli were developed on non-tissue-culture-treated 96-well polyvinyl chloride microtiter dishes (Becton Dickinson, Franklin Lakes, NJ) for 18 h at 30 °C. Thereafter, biofilms were rinsed with diluted nutrient broth (DNB) and 100 μl of harvested predators (~1 × 10⁸ PFU/ml) were added to the wells. Biofilm plates were incubated for up to 48 h. Predator-free DNB was used as a control. The change in biofilm biomass was measured by crystal violet (CV) staining. Experiments were conducted twice with 6 wells for each sample.

3. Results and discussion

In previous studies, we confirmed that the predatory bacteria B. bacteriovorus and M. aeruginosavorus are able to prey on commonly encountered clinical isolates of A. baumannii, E. coli, K. pneumoniae and Pseudomonas spp. which harbor a variety of potent β-lactamases, including extended-spectrum β-lactamase (ESBL), KPC-type carbapenemase, AmpC-type β-lactamase, metallo-β-lactamase, as well as fluoroquinolone-resistant P. aeruginosa ocular isolates [6,15,16]. Although some aspects of the biology of predatory bacteria and what governs prey specificity is not well known, it is believed that attachment of the predators to the prey outer membrane is an essential first step of predation. It was previously reported that mcr-1 encodes phosphoethanolamine transferase, which leads to the addition of phosphoethanolamine moiety on lipid A. This modification reduces the affinity of cationic chemicals such as polymyxins, by altering the negatively charged LPS phosphates [17,18]. This modification in LPS structure was conferred upon the four mcr-1-expressing isolates used in this study [17]. Thus, it could be hypothesized that modification in the prey cell outer membrane in mcr-1 isolates could impact predation and even render the prey resistant. Here we found no change in the ability of B. bacteriovorus to prey upon mcr-1-expressing isolates compared to their colistin-susceptible counterparts. Both B. bacteriovorus 109J and HD100 were able to prey on A. baumannii, E. coli, K. pneumoniae and P. aeruginosa (Table 1). All Bdellovibrio-prey co-cultures exhibited a prey viability reduction of 2.5–5 log₁₀ by 24 h compared to the predator free control. Some differences were seen between the predation ability of B. bacteriovorus 109J and HD100. Most noticeable was the predation measured on P. aeruginosa in which B. bacteriovorus HD100 reduced cell viability by 99% more than the 109J strain. The slight difference in the susceptibility of A. baumannii, E. coli, K. pneumoniae and P. aeruginosa to predation by 109J and the HD100 is in line with previously published reports [6,15] which documented slight differences in predation among the predator strains. Unlike B. bacteriovorus, M. aeruginosavorus exhibited a more limited prey range that included K. pneumoniae and P. aeruginosa. Introduction of mcr-1 into the examined bacteria did not alter predation. For A. baumannii, E. coli, K. pneumoniae and P. aeruginosa, no statistically significant changes in predation were measured in the ability of B. bacteriovorus 109J and HD100 to prey on the colistin resistant isolates as compared to their colistin susceptible counterparts. Furthermore, the introduction of mcr-1 into A. baumannii ATCC 17978 and E. coli ATCC 25922 did not render the transgenic bacteria susceptible to predation by M. aeruginosavorus. The prey range of M. aeruginosavorus demonstrated here is in line with previous prey range analysis done [5,6,15,16], although some E. coli strains are susceptible to predation by M. aeruginosavorus [5]. As the factors impacting prey specificity are not known at this point, we can only speculate on the basis of the inability of M. aeruginosavorus to prey on the E. coli strain used in this study.

Table 1.

Reduction in planktonic prey cell viability (log₁₀ CFU/ml) following 24 or 48 (*) h of incubation.

Prey	Predator
	B. bacteriovorus 109J	B. bacteriovorus HD100	M. aeruginosavorus ARL-13
A. baumannii ATCC 17978	3.17 ± 1.00	3.67 ± 0.32	0.24* ± 0.39
A. baumannii ATCC 17978 pMQ124XLAB1-mcr-1	2.99 ± 0.33	3.71 ± 0.26	0.09* ± 0.12
E. coli ATCC 25922	4.78 ± 0.91	4.56 ± 0.67	0.17* ± 0.30
E. coli ATCC 25922 pMQ124-mcr-1	5.09 ± 0.87	4.90 ± 0.76	0.18* ± 0.18
K. pneumoniae ATCC 13883	3.06 ± 0.46	4.53 ± 0.17	3.40 ± 0.61
K. pneumoniae ATCC 13883 pMQ124-mcr-1	3.66 ± 1.04	4.29 ± 0.92	2.76 ± 0.21
P. aeruginosa ATCC 47085	2.52 ± 0.51	4.53 ± 0.17	2.50* ± 0.47
P. aeruginosa ATCC 47085 pMQ124-mcr-1	3.13 ± 0.12	4.77 ± 0.27	2.14* ± 0.55

Co-cultures were prepared by adding ~1 × 10⁸ CFU/ml prey cells to harvested predator cells (~1 × 10⁸ PFU/ml for B. bacteriovorus and ~1 × 10⁷ PFU/ml for M. aeruginosavorus) or predator free control. Values represent the maximum log₁₀ change as compared to the predator free control. Experiments were conducted twice in triplicate. Values represent the mean and standard error.

In addition to planktonic cells, the ability of predatory bacteria to kill colistin-resistant bacteria in biofilms was analyzed. When pre-developed biofilms were treated for 24 h with B. bacteriovorus 109J and HD100, a 50% and 59% reduction in CV staining was measured on the A. baumannii susceptible strain and the mcr-1-expressing strain, respectively (Fig. 1A). Similarly, no statistical difference was measured in the ability of B. bacteriovorus to remove biofilms composed of the colistin-susceptible and -resistant E. coli isolates (Fig. 1B). As exhibited with planktonic prey cells, no reduction in A. baumannii and E. coli biofilms was measured on biofilms after exposure to M. aeruginosavorus (data not shown). Unfortunately, we were unable to conduct the biofilm removal assay with K. pneumoniae and P. aeruginosa, as the strains used in the study did not form a robust and sustainable biofilm under the tested conditions, to allow the biofilm predation assay to be conducted.

Fig. 1.

Removal of biofilms by B. bacteriovorus. Biofilms of A. baumannii (A) and E. coli (B) were developed in 96-well plates for 24 h (preformed biofilm) following a 24 h incubation period with B. bacteriovorus 109J (109J), B. bacteriovorus HD100 (HD100), or predator free control (control). Biofilms were rinsed and stained with CV and the amount of CV staining was quantified at 600 nm (A₆₀₀). Each value represents the mean of 12 wells. Error bars are shown as one-standard deviation. Numbers above the bars represent the average percent reduction in biofilm biomass compared to the control.

In conclusion, in this study we have established that predatory bacteria are able to prey on Gram-negative colistin-resistant isolates expressing mcr-1. The work highlights the potential application of predatory bacteria as a living antimicrobial to be used to treat untreatable antibiotic resistant pathogens in the future.