Sunday, September 17, 2017

Compound isolated from C difficile act against C difficile: Avidocin-CDs


Clostridium difficile represents a unique problem in health care system. C difficile is not affected by a wide range of antibiotics and they start showing up when gut microbiome is depleted due to antibiotics. C difficile is often treated with antibiotics such as vancomycin, metronidazole and fidaxomicin which cause further disruption of the resident microbiota leading thus increasing chances of relapse. A faecal microbiome transplant (FTM) is an option but it comes with some challenges. For example, It is nearly impossible to rule out every possible infection from a healthy donor and we are technologically incompetent yet to find the right best combination of microbiome for the gut. The best answer would be an antibiotic that kills only and only C diff. That means we have to be extremely specific.

Fig 1: Electron micrograph of negatively stained
purified diffocin particles isolated from CD4 strain.
C difficile is a highly competitive bacteria. C difficile strains compete with each other and they produce an R-type bacteriocin (High-molecular-weight or phage tail-like) which is called as diffocins. In a generalised sense, R-type bacteriocins attack target cells by specific receptor-binding on the surface. This is followed by sheath contraction and insertion of the core through the envelope of the target bacterium. The diffocins are ultra specific in their activity and their receptor binding activity can be modified to attack C difficile in general by modifying its binding to specific receptors. 

Table 1: Sensitivity of various bacteria to modified diffocins.
Source
Table 1 shows a summary of the sensitivity of various bacterial strains to heterologously expressed, recombinant diffocins. A company that is currently working on using these diffocins and their modified version- Avidbiotics Corp (California based Biotech company) have named them as Avidocin-CDs. One of such constructs Av-CD291.2 had been found to have wide spectrum activity against a range of hypervirulent C difficile strains tested, without affecting the microbiome.

There is sufficient literature evidence to indicate that avidocins are highly specific for C difficile and able to survive transit through mouse GI tract.

In the latest paper, the story becomes better now. The team of scientists from the University of Sheffield, AvidBiotics Corp, and the University of Glasgow have published a study on Avidocin-CD291.2 on how it could be extended for clinical use. Analysis of rare Av-CD291.2–resistant mutants enabled identification of S-layer protein A (SlpA) as the target. The paper showed that Av-CD291.2–resistant mutants lack an S-layer. The lack of S-layer also introduces a high sensitivity to innate immune molecules combined with sporulation defects. These S-layer mutant strains survived poorly in the standard charcoal medium which is used to transport C difficile strains. Interestingly, acquisition of Avidocin-CD resistance results in loss of toxin production and complete loss of virulence.

As Dr Robert P. Fagan, senior corresponding author comments, "We discovered that the weapons naturally produced by C difficile and those engineered by our colleagues at AvidBiotics were using certain proteins in the S-layer to identify which strains to target. The C difficile S-layer is unique to these bacteria, which explains why Avidocin-CD killing is so specific. Scientists at AvidBiotics Corp were then able to engineer different versions of Avidocin-CD to target 12 of the 14 known types of S-layer."

Reference:

Joseph A. Kirk, Dana Gebhart, Anthony M. Buckley, Stephen Lok, Dean Scholl, Gillian R. Douce, Gregory R. Govoni, Robert P. Fagan. New class of precision antimicrobials redefines role of Clostridium difficile S-layer in virulence and viability. Science Translational Medicine, 2017; 9 (406): eaah6813 DOI: 10.1126/scitranslmed.aah6813

Tuesday, September 05, 2017

Hypervirulent Klebsiella pneumoniae

One of the most common ideas associated with clinical antibiotic resistance is the fitness factor. I have in multiple posts talking about this idea in my previous posts. The idea is that when a bacteria acquired genes for antibiotic resistance there is a fitness cost associated with it. Until and unless there is this constant antibiotic threat to the bacteria, it is not so useful for the bacteria to keep resistance genes, since there is a cost associated with maintaining that gene. There are several experiments that showed at least for some drugs that if you mix a sensitive and resistant phenotype of a given organism and allow it grow together on an antibiotic free condition, the sensitive strain tends to dominate easily.

The idea is all fine and good in laboratory conditions. In a series of 5 hospital acquired pneumoniae infection (All fatal) following surgery in a Chinese hospital, a Klebsiella pneuomoniae ST11 strain has been isolated which is not only an MDR (Multi drug resistant) but also a hypervirulent strain.

The study, in summary, basically went something like this. There were 5 back to back pneumoniae cases following surgery for traumatic cases who developed pneumoniae and died of it. The first one in the series was identified as the most probable patient zero (Index case). The other four patients were located in different wards with overlapping stays. In all the cases multiple samples were taken and all the Klebsiella pneumoniae strains isolated were fully characterised using phenotypic and genetic approaches. In total 21 non-repeated carbapenem-resistant K pneumoniae strains were recovered from various clinical specimens of the five patients

What was really interesting to me was that they did a string test, human neutrophil assay and a wax moth virulence test to establish its hypervirulence status. These tests are really valuable models and so I think I should explain these in a little bit of detail.

Photo 1: Positive “String test” on a hypervirulent
strain of K. pneumoniae. Source
String test (Don't confuse this with the string test done to identify Vibrio cholerae), is a kind of qualitative marker used to test the hypermucoviscous phenotype seen in Klebsiella pneumoniae which are an indicator or hypervirulence. Basically, you grow the organism on a 5% sheep blood agar at 37°C overnight. The string test is positive when a bacteriology inoculation loop or needle is able to generate a viscous string > 5 mm in length by stretching bacterial colonies on an agar plate. See Photo 1. In this paper, authors reported that the string test was positive for all five strains, each producing strings longer than 20 mm.

Fig 2: Neutrophil Survival of K pneumoniae strains.
Source
Neutrophil assay is a test to see how good the bacteria resist killing by the neutrophil in laboratory conditions. In principle,  neutrophils obtained from healthy volunteers. A well containing 10⁶ neutrophils and 10⁶ CFU of opsonised K pneumoniae in RPMI/H medium is prepared at 37°C. A small sample is taken at intervals and bacteria is plated on Luria broth agar. Survival is calculated as a percentage of CFUs with reference to controls. For the study, K pneumoniae 4 and 5 (representative strains), two classic ST11 strains FJ8 and FJ9 (known to be not hypervirulent strains) and two known K1 hypervirulent K pneumoniae strains 1088 and 91 along with PC K pneumoniae which was removed for its virulence plasmid were studied. Fig 1 shows the results. You can clearly see that the strains 4 and 5 were really evading the neutrophil killing.

Fig 2: Virulence potential of isolated strains.
Source
Another test for virulence was done using Galleria mellonella or honeycomb moth. Basically, the bacteria are incubated with the larvae and you look for the ability of the larva to survive. For this study, cultures of K pneumoniae strains were washed with PBS and larva was infected with the bacteria and survival rate of the larvae was studied. See Fig 2. 

The study does provide a compelling evidence that the strains they isolated were hypervirulent. Yes, they were also MDR strains but most of the K pneumoniae strains isolated globally are MDRs. But mind you they are not super bugs. I wasn't sure if these strains were resistant to drugs like Colistin and from the data they appear tigecycline sensitive.

I take this study as a proof that ST11 type which is a common circulating type in Asia is capable of hypervirulence. They have a 170 kbp plasmid (pVir- CR-HvKP4) which makes it hypervirulent, multidrug resistant, and transmissible. Considering so many people are dying from Klebsiella pneumoniae infections it may be worthwhile to test how many percentages of it is actually hypervirulent.

Reference

Danxia Gu, Ning Dong, Zhiwei Zheng, Di Lin, Man Huang, Lihua Wang, Edward Wai-Chi Chan, Lingbin Shu, Jiang Yu, Rong Zhang, Sheng Chen. A fatal outbreak of ST11 carbapenem-resistant hypervirulent Klebsiella pneumoniae in a Chinese hospital: a molecular epidemiological study. The Lancet Infectious Diseases, 2017. https://doi.org/10.1016/S1473-3099(17)30489-9