Friday, May 26, 2017

Understanding Anelloviruses

Recall that I have recently written a post on human blood virome and one of the major findings was that there are a lot of viruses in human blood circulation that is apparently normal. A major critique of the study is that there are a lot of contamination issues to be ruled out. It is really not clear as to if the virus is actually in the blood, or has been introduced from the skin during the collection procedure. If you look at the past literature on WGS technology it is undisputedly clear that in most cases skin microbiome is introduced during blood collection even when sterile precautions are taken, at a non-negligible level. One virus that was particularly striking was Anellovirus, something not many people have heard about. That is the point of discussion here.

Photo: Torque teno virus.
Anellovirses are non-enveloped, icosahedral symmetry virus (T=1) with a genome containing a circular single-stranded DNA coding for 3 major open reading frames (ORFs). In addition, they carry several small ORFs and untranslated regions (See Fig 1). Torque teno virus (TTV), is the most studied member of the group which was first reported in 1997 in a Japanese patient post-transfusion. 2 more virus that is now commonly talked about includes Torque teno mini virus (TTMV) and Torque teno midi virus (TTMDV). Subsequently, there have been several different studies on Anellovirus in blood and blood derived products. In all of the studies, Anelloviruses have been found in varying percentages.

Fig 1: Genome organization of
Torque teno virus. Source
It is my understanding that Anelloviruses was previously described as under the family Circoviridae, genus Anellovirus. Currently, Anellovirus itself is the family and have several genera under them. See Table 1 for a compiled information on the classification details.

Anellovirus infection though earlier thought to be more of blood-related have been identified as ubiquitous and estimates are that as much as 90% of global population harbours Anellovirus. This can be seen by detecting the sequences in at least one of the biological samples such as blood, skin, saliva, gut etc. Further, these are not human specific and Anelloviruses have been found in other species such as human primates and wide variety of domestic animals. 

Table 1: Anellovirus classification.
Anelloviruses have been implicated as a correlation with conditions such as fever and associated with hepatitis virus infections. However, they appear to be more of a random phenomenon and no study has been published implicating them as a significant contributor and modulator of a clinical condition. For the time being at least, they appear to be floaters (Commensals) and nothing much of their biology is known.

References

1. McElvania TeKippe E, Wylie K, Deych E, Sodergren E, Weinstock G, Storch G. Increased Prevalence of Anellovirus in Pediatric Patients with Fever. PLoS ONE. 2012;7(11):e50937. 

2. Al-Qahtani A, Alabsi E, AbuOdeh R, Thalib L, Nasrallah G. Prevalence of anelloviruses (TTV, TTMDV, and TTMV) in healthy blood donors and in patients infected with HBV or HCV in Qatar. Virology Journal. 2016;13(1).

3. Spandole S, Cimponeriu D, Berca LM, Mihăescu G. Human anelloviruses: an update of molecular, epidemiological and clinical aspects. Arch Virol. 2015 Apr;160(4):893-908

Wednesday, May 17, 2017

VPM1002 TB vaccine to be tested in India

Tuberculosis continues to be a high burden problem in many parts of the world especially, its co-infection with HIV creates substantial complications. Though BCG is universally administered, BCG fails to protect after a certain number of years. Research is currently focussed on inventing a totally new vaccine or to create modifications in BCG allowing better vaccine performance. In India, a recombinant vaccine called VPM1002 is planned to be tested for phase II/III vaccine trial. Here are some details of this vaccine.

Fig 1: Schematic description of the underlying
mechanism of improved T cell stimulation
by VPM1002. Source
BCG is derived from M bovis and has immunological properties which is much similar to M tuberculosis. The bacteria is phagocytosed by host macrophages, and are efficiently trapped in cellular phagosome. However, the cell fails to digest the bacteria due to multiple virulence mechanism of the bacteria. One of them is a urease which plays a role in pH neutralization of phagosome thus denying its maturationSubsequently, antigens are processed by MHC II pathway and induce CD4+ T-cell responses but what is actually required is a CD8+T cell response. In 2005 a JCI paper reported a rBCG (Recombinant BCG) prague strain that secretes listeriolysin of Listeria monocytogenes and is urease C-deficient. The idea being that the a urease deficient mycobacterium secreting listeriolysin allows better phagosome maturation and potentially activating MHC I pathway thus eliciting a desired CD8 response. Vaccine developed from this strain is called as VPM1002. Prof. Kaufmann comments “The vaccine being tested is intended to replace the current BCG vaccine and will be administered to young children to protect them against tuberculosis. Adults may also be able to benefit from it later”. In a phase I Clinical trial conducted in Germany  and and South Africa, volunteers were followed up for 6 months after a single vaccination with 5 x 10 CFU was safe, well tolerated and induced multifunctional CD4+ and CD8+ T-cells. Studies have also showed that rBCG is significantly better in terms of immunity and safety. The findings were subsequently confirmed by a phase 2a study in South Africa.

Photo: VPM1002 Vaccine.
Source
India being a high burden for TB desperately needs a new and better vaccine. Serum Institute of India has apparently obtained approval to go ahead with Phase II/III vaccine testing. This will be a double blind placebo-controlled, randomised clinical trial which will see enrolment of 2,000 adults (1000 participants will receive vaccine and the other 1000 will receive placebo) in 17 centres. A single dose will be administered and followed up for 12 months. The work is planned in 2 phases. In the 1st phase, 200 participants will be given the vaccine and safety evaluated. In the second phase (given the success of first phase), the remaining volunteers will be vaccinated. Dr. Prasad S. Kulkarni, Medical Director at Serum Institute says, “Adults who have completed TB treatment will be first screened and enrolled if found eligible 2-4 weeks after completion of TB treatment. Traces of the drugs may be present in the body for two weeks after completion of the treatment. Since the vaccine contains live, weakened bacteria, the drugs can kill them if given earlier than two weeks after completing the treatment.”

The key importance of VPM1002 in contrast with BCG is superior immunity conferred by CD8+ T cells, enhanced Il-17 secretion. It also reduces the risk associated with BCG complications seen in a small subgroup of HIV positive subjects.

References:

Grode L. Increased vaccine efficacy against tuberculosis of recombinant Mycobacterium bovis bacille Calmette-Guerin mutants that secrete listeriolysin. Journal of Clinical Investigation. 2005;115(9):2472-2479.

Grode L, Ganoza C, Brohm C, Weiner J, Eisele B, Kaufmann S. Safety and immunogenicity of the recombinant BCG vaccine VPM1002 in a phase 1 open-label randomized clinical trial. Vaccine. 2013;31(9):1340-1348.

VPM Study group. HIV-unexposed newborn infants in South Africa in HIV-unexposed newborn infants in South Africa. Clin. Vaccine Immunol. doi:10.1128/CVI.00439-16

Tuesday, May 09, 2017

Atlas Map for blood Dendritic cells, Monocytes, and Progenitors

I note that I have been unable to post frequently in past few weeks, since I have been very busy with work and personal travel. I have been getting a few mails enquiring if I moved to a new page. No, I havent yet. If I do, I will definetly make an announcement.

Immunology is still a very difficult subject especially when it comes to classification. The problem arises from the lack of understanding and partly cause classification system is messy. Further, the same immune cell function differently under different circumstances. Cellular subtyping and subsequent studies are based largely on the established classification rules, which has been developed decades ago. In most cases, a subtypes is based on morphology and a little understanding of function. For most of the human cells, there is no clear cut systematic cellular classification based on molecular profile. Human cell atlas in one of the ambitious project that has been announced to overcome this massive question. Sarah Teichmann states, “The cell is the key to understanding the biology of health and disease, but we are currently limited in our understanding of how cells differ across each organ, or even how many cell types there are in the body. The Human Cell Atlas initiative is the beginning of a new era of cellular understanding as we will discover new cell types, find how cells change across time, during development and disease, and gain a better understanding of biology.”

The methodology used for this is technically complex but the idea is straightforward. The cells are sorted out using a known classification system and then each cell is isolated from the pool. Then using a technique called single cell transcriptomics a cell is profiled. This gives a picture of what is the genetic expression pattern of that particular cell. By parallely doing so for a huge number of cell samples, a map can be drawn.

Dendritic cells are called so because of their structure. They are one of the most important classic APC (Antigen presenting cell). Dendritic cells (DC) were first discovered by Ralph Steinman roughly 4 decades ago for which he was awarded the Nobel prize. Though there has been some previous hints that DCs are a diverse population, the subtypes have not been recognised or typable. 


In the study by Villani et al human PBMCs were enriched from blood sample using Ficoll extraction and DCs were isolated using flow cytometry. Each cell was then isolated into a single well and transcriptome analysis was done. For the study, 742 DCs and 339 monocytes that passed quality control was profiled with an average detection of 5326 genes per cell. Based on genetic expression cluster results 6 clusters which are numbered DC1 to DC6 were identified (See Fig 1 for classification details).
  • 2 clusters mapping closely to the well-established DC subsets, with cluster DC1 mapping to CD141+ DCs and cluster DC6 to pDCs.
  • 2 clusters containing the CD1C+ cDCs, cluster DC2 and cluster DC3
  • 1 cluster corresponding to CD141–CD1C– population named DC4
  • 1 cluster DC5
Fig 1: Summary of the study and revised classification of the Dendritic cells to include 6 different subtypes based on the transcriptomic map. Source
Further analysis was able to gathered some potential markers of interest.
  • DC1 corresponds to CD141/BDCA-3+ ,which was best indentified using CLEC9A.
  • DC2 and DC3 togther corresponds with CD1C/BDCA-1+
  • DC4 corresponded with CD1C–CD141–CD11C+ which also shared some signatures with monocytes
  • DC5 was unique in itself.
  • DC6 corresponded to the interferon-producing pDC, defined by standard markers CD123, CD303/BDCA-2
The paper is very dense with lot of data. However, essentially this paper established that there are DC subtypes defined and probably there is a diversity in function. This also sits well with literature where function of DCs is called into into three categories. First, antigen presentation and activation of T cells. Second, inducing and maintaining immune tolerance. Third, maintaintainence of immune memory. However, which does what is yet to be studied and elucidated. Divya Shah comments, “In this study, scientists have used cutting-edge technologies to find that there are many more types of cell than we originally thought. The next step is to find out what each of these cell types do in our immune system, both when we’re healthy and during disease."

Reference

Villani A, Satija R, Reynolds G, Sarkizova S, Shekhar K, Fletcher J et al. Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors. Science. 2017; 356 (6335): eaah4573.

Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors

Monday, April 24, 2017

Lab Series #16: Anti Nuclear antibodies

Occasionally in this blog, I write about immunology topics. But I have rarely discussed diagnostic immunology in this blog. Of course, I have written a post long back on how immunology-based methods are used for diagnostics but not really anything about diagnosing an immunological disorder. Autoimmune diseases are a huge topic to discuss and write about. Ask a rheumatologist, there are hundreds of different types of them. To talk about everything is a huge task (I will have to start writing a new blog for that!!). Instead, I want to focus on a common test, casually called as ANA- Global and Profiling tests. I specifically chose this topic since there are some particular confusions that many don’t clearly understand about.

Photo 1: LE cell. Source
In 1948, the observation of LE cells in a patient with SLE was a key realization that autoimmune reactions are a significant clinical condition. This discovery lead to more discoveries on characterization of antibodies directed against nuclear antigens what is now designated as “ANA or anti-nuclear antibodies”. An LE cell is a neutrophil or macrophage that has phagocytized the denatured nuclear material of another cell. For a long time, LE cell was used as a diagnostic methodology. The principle is very simple. Make a buffy coat preparation from patient blood sample and break some cells such that the nucleus is exposed. Make a smear of the preparation. If ANA is present, this will trigger phagocytosis of nuclear material and can be seen as an LE cell. A smear is considered positive when 10 or more characteristic LE cells are seen in a smear preparation (See Photo 1). The test is laborious and have some demerits. In modern day practice, we no more do this test. Instead, we screen for ANA by a screening test using indirect Immunofluorescence. The screening test is called ANA global and identifying the antibody is called as ANA profiling.

George Friou was the first who applied the immunofluorescence technique for the detection of ANA. In the later years, different immunofluorescence patterns of ANA were observed and lead to the realisation that there are several different possible ANA. By the 1970’s the indirect immunofluorescence test was established using tissue sections as substrate. By 1980’s HEp-2 (Human epithelial cell line) was established as a substrate for ANA screening. Currently, Immunofluorescence testing method is considered as a gold standard for diagnosis. 

So, what makes a Hep-2 cell the standard? HEp-2 cells are laboratory maintained cells (derived from human laryngeal carcinoma) and hence it brings in the uniformity of testing. But a more important reason is these cells are neoplastic in nature and have a large nucleus which gives an advantage in viewing the results. The methodology of ANA- IF testing is very simple. The exact details differ between manufacturers kits though the underlying idea is uniformly the same. Patient plasma/serum sample is overlaid on the HEp-2 cell line. If auto antibodies are present it binds to the antigens in the cell whcih is detected using a secondary antibody. A negative Immunofluorescence (IF) result is a confirmation of negative for autoimmune antibodies.

Interpretation of the findings is based on 3 different parameters.

  • Fluorescence pattern
  • Substrate tissue
  • Sample dilution (titer)
Fluorescence pattern:

There is a huge number of possible patterns recorded in literature that can be seen in the IF testing. Though technically speaking the test is specific for nuclear antibodies, the substrate in use is a complete cell and hence if there are autoantibodies to antigens other than the nuclear antigens you are bound to see those patterns too. There are 3 basic possible patterns that can be seen in an ANA-IF global testing. See Fig 1, for some basic idea of classification.

Fig 1: Common immunofluorescence patterns.
HEp-2 cells, as already stated are neoplastic cells. Most of the cells have a higher nuclear DNA content than you would see in a normal cell and rate of cell division is higher. Hence you will see in any given HEp-2 cell line coated with a slide, a number of cells that are in interphase and a good number of cell in metaphase. This is advantageous cause there are certain patterns better visualised in interphase cells and certain patterns in metaphase cells.

A good number of people interpret the results just by looking into a couple of fields. Though it is time-saving, it is sometimes beneficial in checking more than a few fields under a microscope. Another common mistake is to interpret the positivity of test by fluorescence intensity alone. Washing step after labelling is a crucial step and sometimes despite good and skilled hands, there tends to be a low background fluorescence. This is often referred to as background noise. Sometimes, this fluorescence can be stronger and relying too much on fluorescence maybe misleading to falsely identify a negative as low positive. The best method is to follow a rule - If you see the same pattern all over the slide (in any part of the preparation you observe) and if you can clearly differentiate nucleus and cytoplasm by fluorescence, with a specific pattern only then it is a positive.
Fig 2: Most common nuclear patterns observed under fluorescence microscopy. Source

Substrate tissue:

As already discussed above, substrate tissue determines what antigens you are looking at. ANA kits though are ideally made to detect only ANA, the occurrence of other auto-antibodies has prompted to include more cell lines in a single assay (Example: Euroimmun biochip). Certain antibodies are neagtive on mouse kidney or rat liver but are positive on HEp-2 cell line. For most purposes, ANA is considered classically on HEp-2 cells. 


ANA titer:


The dilution of the sample at which the fluorescence is positive is an indicator of the quantity of the auto antibody. There is a lack of consensus on what should be the minimum titer for a sample to be declared as positive. This is because a good number of normal population is positive for ANA at lower titers. For example, at a dilution of 1:40 roughly 20% of the population is positive. This percentage changes depending on location, Age, Sex etc. By increasing the titer to 1:160 this positive rate comes down to about 3%. A good number of kits now recommend 1:160 as an optimal titer. It should be noted that there is a still a small amount of false positive rate.

Table 1: ANA Profiling tests for detection of specific antibodies.
Source
A global assay (Generic assay) is a screening test. It cannot predict the autoantibody. For example, a homogenous pattern indicates antibody against ds- DNA, histone or nucleosome. So when an ANA global is positive, a profile is ordered.

In the earlier days, antigen preparation was not advanced. Only a handful of the antigens could be purified in the laboratory. These antigens that could be extracted are called as ENA (Extractable Nuclear antigen). Some of the common known ENA's include dS-DNA, Histone, Centromere, JO-1, LA(SS-B), RNP, RO (SS-A), SCL, Sm etc. Today more and more antigens are purified or available in a pure form to test. There are several methods available in current practice for profiling the ANA. See Table 1. Fig 3, shows an example multiparametric immunoblot from Euroimmun for differentiation and confirmation of the 23 ANA in a single test run.

Fig 3: ANA 23 profile. Source

The ICAP (international consensus on antinuclear antibody pattern) has renamed the nomenclature for reporting ANA. Instead of name, each pattern is assigned an AC code. For example, the homogenous pattern will be called as AC-1 (Read as Anti-cell pattern-1). Also, they have categorised the patterns into competent level reporting and expert level reporting based on clinical relevance and easiness of identification. The requirement is that the patterns under competent level should be reported and expert level reporting is optional.

Fig 4: Nomenclature and Classification Tree. Source
References

Kumar Y, Bhatia A, Minz R. Antinuclear antibodies and their detection methods in diagnosis of connective tissue diseases: a journey revisited. Diagnostic Pathology. 2009;4(1):1 DOI: 10.1186/1746-1596-4-1

Chan Ek et al. Report of the First International Consensus on Standardized Nomenclature of AntinuclearAntibody HEp-2 Cell Patterns 2014-2015. Front Immunol. 2015 Aug 20;6:412. doi: 10.3389/fimmu.2015.00412.

The official website for the International Consensus on Antinuclear Antibody (ANA) Pattern (ICAP). http://anapatterns.org

Friday, March 31, 2017

Human Blood Virome

There is no denying that blood transfusion is an important part of modern medicine. In a good number of cases, the whole blood need not be given but rather a component of blood can be given. This allows the same blood to be given as components to different recipients as required. The ability to transfuse blood or its components is lifesaving in most cases. Bloodborne infections such as HIV, Hepatitis B and C etc. are routinely tested in the lab before the transfusion. As per the current guidelines, blood and blood-derived products (BBDP) are tested for a set of microbial agents which are thought to be pathogenic and transfusion- transfusible.

There is a debate on if we are testing enough. For example, testing strategies currently use an immunology-based assay for detection of signatures related to infections and that is not as sensitive as genetic tests. There are many other region specific infections such as Ebola and Zika virus (especially since certain carriers are asymptomatic) which can be transmitted by blood for which routine testing is not done. We are not even aware of every possible range of organisms that can be transmitted, let alone test. In an ideal case, the BBDP should be free of any microbial component. However, such a scenario is unlikely. Just as many other body fluids have been shown to be non-sterile, blood is also shown to have a good number of microbiome associated with it.

Most of the microbiome papers are actually bacteriome papers. Bacteriome papers are much more common in literature since it involves sequencing of 16s rRNA and it is much easier to do. In similar lines, mycobiome can be done, since it involves sequencing a particular target. In contrast, there is no common targetable gene or region of the genome for viruses and the only viable approach for studying virome is to do whole genome sequencing. Human virome has been published heavily for skin and gut and the major findings have been bacteriophages of various types. Blood virome is thus an interesting study that looks into what viruses are there in blood and are we testing enough.

In 2003, a proposal was made to develop a global system to catalog viruses and detect emerging diseases throughout the world. The idea was to routinely screen human blood to identify and monitor viruses from human samples.  The core of the proposal was to collect blood from hospitals and labs weekly. This would be followed by extraction and sequencing of the viral genomes. Once a database of viruses is constructed, researchers could use it to screen for new viruses as they appear in the population. Such a database was supposed to help in quick identification of emerging infections and identify novel viruses.

A multi-group collaboration involving Dr. J Craig Venter (recipient of De Leeuwenhoek Medal 2015) who has been recognized for developing several generic findings by sequencing technologies has now published a paper on human blood virome from Human Longevity Inc. To put the study in a nutshell they sequenced genomes of 8,240 individuals who were all essentially healthy and not infected with anything. The majority of the reads were mapped to the human reference genome. Roughly 0.2% was attributed to bacteria and 0.01% to viruses. Fig 1 shows a summary of the percentage of individuals presenting with viral sequences. It was not surprising to see a variety of human herpes virus, but there were many other viruses some of which are related to humans and many bacteriophages. It must be noted that this study was designed to look for DNA viruses and there would be much more in the data if RNA-related virome reads can be obtained. 

Fig 1: Prevalence and abundance of human DNA viruses and retroviruses in 8,240 individuals. A) Frequency B) The viral load. Source
The study is important since it kind of establishes what is a normal virome. Novel genomes were not reported in the paper. As the paper concludes "The study was not conceived for the discovery of highly divergent, novel human viruses, as this requires the use of less stringent similarity criteria for detecting divergent (relative to those already known) viral sequences."

Reference

Moustafa A, Xie C, Kirkness E, Biggs W, Wong E, Turpaz Y et al. The blood DNA virome in 8,000 humans. PLOS Pathogens. 2017;13(3):e1006292.

Wednesday, March 22, 2017

MCR gene variants

Table 1: Known mechanisms of colistin resistance.
MCR-1 a plasmid mediated resistance gene has been extensively discussed on this blog previously. Ever since its first report, a lot of research papers have come from the topic. There is hardly a country where genomic surveillance tools are available and MCR-1 is not reported. The location of the gene on a plasmid makes it more devastating. It is indeed very interesting to note that the MCR-1 is not just located in one plasmid. Many different plasmids have been demonstrated to be capable of harbouring MCR-1 and hence it should be inferred that it is widely distributed. It should be noted that the colistin resistance can be caused by several factors. Table 1 is a compiled list of colistin resistance determinants.

Fig 1: Structure of the catalytic domain of MCR-1
phosphoethanolamine transferase. Source
The crystal structure of MCR-1 is already published and a good lot of information is available about the domain. The crystal structure study showed that MCR-1 possess a α-β-α-sandwiched structure and coordinate divalent zinc ions in the active site via phosphorylation of the conserved residue threonine. The nucleophile for catalysis is threonine 285 (Phosphorylated). They are most homologous to LptA and EptC transferases in terms of the sequence.

What is most interesting is that MCR variants are now being described. In a surveillance study at Belgium, 92 porcine and bovine colistin-resistant E coli isolates that were negative for MCR-1 was identified. Of these randomly selected 10 isolates, plasmid sequencing was done and 3 of the 10 showed sequence later designated as MCR-2 gene, which was 1,617 bp long, nine bases shorter than mcr-1(1,626 bp), and showed 76.75% identity. In 2016 august, a group from Italy described a novel MCR variant, named MCR-1.2, from an MDR KPC-producing K pneumoniae strain belonging to sequence type 512. This strain (KP-6884) was isolated in 2014 from a rectal surveillance swab obtained from an Italian child admitted to the paediatric onco hematology ward of Pisa University Hospital.

Fig 2: Sequence comparison of MCR and its variants.
Source
Most recently another MCR-1 gene variant, referred as MCR-1.3 carried is reported from a colistin-resistant S. Typhimurium strain YL14P053, which was isolated in 2014 from a rectal swab from a 46 years old healthy woman who received a medical examination in the Yulin Center for Disease Control and Prevention. It is a little confusing to me that the news and press release article mentions this variant as an MCR 1.6 whereas in the paper it says MCR 1.3. Fig 2 shows the alignment of MCR-1 gene with its variants are shown. MCR-1.2 has a variation at the nucleotide position 8, A was mutated to T, leading to a Gln to Leu change in amino acid position 3; MCR-1.3 has a variation in nucleotide position 1263, G was mutated to A, which is a synonymous mutation, while in nucleotide position 1607, G was mutated to A, leading to an Arg to His change in amino acid position 536.

Corresponding author Biao Kan comments, "This is the first time a mcr-1 gene has been found in Salmonella in a healthy carrier. Healthy carriers play an important role in the transmission of resistance genes to the community. Salmonella infections have been the leading cause of foodborne illness, and Salmonella-carrying mcr-1 will likely be a problem in food safety".

It occurs to me that the MCR gene and its variants have been found in several scenarios where there is no colistin pressure. That means evolutionarily there is no great fitness cost in keeping it, further suggesting that losing this plasmid would be a common feature once acquired.

Just like there are several NDM types and variants after the original discovery of NDM-1, am sure we are going to have several types of MCR-2. If we start whole genome sequencing of every colistin resistant isolate we are going to definitely get a lot of variants and maybe more types. Or who knows, something totally novel.

References

Olaitan A, Morand S, Rolain J. Mechanisms of polymyxin resistance: acquired and intrinsic resistance in bacteria. Frontiers in Microbiology. 2014;5. 

Ye H, Li Y, Li Z, Gao R, Zhang H, Wen R et al. Diversifiedmcr-1-Harbouring Plasmid Reservoirs Confer Resistance to Colistin in Human Gut Microbiota. mBio. 2016;7(2):e00177-16. 

Stojanoski V, Sankaran B, Prasad B, Poirel L, Nordmann P, Palzkill T. Structure of the catalytic domain of the colistin resistance enzyme MCR-1. BMC Biology. 2016;14(1). 

Hu M, Guo J, Cheng Q, Yang Z, Chan E, Chen S et al. Crystal Structure of Escherichia coli originated MCR-1, a phosphoethanolamine transferase for Colistin Resistance. Scientific Reports. 2016;6(1).

Xavier BB, Lammens C, Ruhal R, Kumar-Singh S, Butaye P, Goossens H, Malhotra-Kumar S. Identification of a novel plasmid-mediated colistin-resistance gene, MCR- 2, in Escherichia coli, Belgium, June 2016. Euro Surveill. 2016;21(27):pii=30280

Di Pilato V, Arena F, Tascini C, Cannatelli A, Henrici De Angelis L, Fortunato S et al. mcr-1.2, a New mcr Variant Carried on a Transferable Plasmid from a Colistin-Resistant KPC Carbapenemase-Producing Klebsiella pneumoniae Strain of Sequence Type 512. Antimicrobial Agents and Chemotherapy. 2016;60(9):5612-5615.

Lu X, Hu Y, Luo M, Zhou H, Wang X, Du Y et al. MCR-1.3: a new MCR variant carried by an IncP plasmid in a colistin-resistant Salmonella enterica serovar Typhimurium isolated from a healthy individual. Antimicrobial Agents and Chemotherapy. 2017;:AAC.02632-16.

Friday, March 17, 2017

Bedaquline and Delamanid resistance

Greetings,

Fig 1: Bedaquiline marketed as Sirturo.
Source
Recently, I attended a talk on tuberculosis by Soumya Swaminathan who covered a great in depth detail on current scenario of tuberculosis. During the talk, it just occurred to me that bedaquiline and delamanid has been already approved for use in many different countries. Considering that the experiments published for assessment of the drug target had mutations, it was kind of striking to me think that bedaquiline resistance is already there. Though I have read some reports here and there, I never gave this whole idea a detailed look. Bedaquiline is a new generation anti TB drug markerted by the trade name of Sirturo. Bedaquiline works by blocking an enzyme inside the Mycobacterium tuberculosis bacteria called ATP synthase. The specific loss of ATP synthase activity basically kills the bacteria. A detailed pharmocological profile and other related details can be found here.

In an article published in NEJM by Bloemberg etal;2015 a case of is recorded where the patient showed up with TB and repeatedly acquired resistance to multiple antibiotics including bedaquiline and delamanid. As the resistance developed, the genome was sequenced which showed mutation in mmpR that was associated with bedaquiline resistance. Subsequently when Delamanid was started mutations in fbiA and fgd1 developed asssocaited with resistance to delamanid. Several reports are published where bedaquiline resistant strain have been sequenced and genes associated with resistance that were found include upregulation of MmpL5 (multisubstrate efflux pump), AtpE gene mutations and pepQ mutation. Interestingly, many mutations that effect bedaquline resistance also gives a cross resistance to Clofazimine. Bedaquiline-resistant mutants arise at a rate of 1 in 108Delamanid is a drug that has come after bedaquiline, but there are now reports of resistance for that too.

Currently, bedaquline and delamanid are on the final frontiers of treating XDR TB cases. It must be noted that the Bedaquiline and Delamanid are not yet fully available in all countries and most places these are used as testing drug. But popping up of resistance already is an indication that once widely available resistance is going to be really common. Some countries including India, have made it mandate that these drugs are not available over the counter and can be obtained only in governemnt supported RNTCP clinics. This greatly reduces the risk since these drugs are given only in very specific conditions. Considering the rising number of resistance in TB to first line drugs am sure that we are looking into a future where TB is mostly treated with second or third line of anti-TB drugs. That means, there is a desperate requirement for anti TB drugs.

Sutezolid, an oxazolidinone is one such candidate currently in development as a treatment for extensively drug-resistant tuberculosis. TBA-354 (A Nitroimidazole derivative) is another drug for which there is great hopes vested. There is also some research happening in the field of mycobacteriophages, but its clinical application is on a very long way.

References:

1. Andries K, Villellas C, Coeck N, Thys K, Gevers T, Vranckx L et al. Acquired Resistance of Mycobacterium tuberculosis to Bedaquiline. PLoS ONE. 2014;9(7):e102135. 

2. Acquired Resistance to Bedaquiline and Delamanid in Therapy for Tuberculosis. New England Journal of Medicine. 2015;373(25):e29-e29.

3. Hatfull G. Mycobacteriophages: Windows into Tuberculosis. PLoS Pathogens. 2014;10(3):e1003953.

Tuesday, March 07, 2017

WHO global PPL for antibiotic development

I have previously mentioned in my several earlier posts that there is a growing concern for antibiotic reistance and new antibiotics needs to be brought into clinical picture. The picture is complicated by several companies not investing sufficiently on R&D for antibiotic discovery and bringing them into clinic. Antibiotics are something that has to be used with care and caution. There are examples of bacteria that are quick to evolve and develop resistance and there are a set of organisms that are not so. Also, there are multiple infections that are not life threatening and can be managed more easily in comparison to certain others that reuqire very aggressive treatment.

From a marketing strategy point, R&D devoted to antibiotic discovery is based largely on companies own decisions which is based on multiple opinions. In other words there are no clear global guidelines on what needs to be really looked into. As Dr. Marie-Paule Kieny comments, "Antibiotic resistance is growing, and we are fast running out of treatment options. If we leave it to market forces alone, the new antibiotics we most urgently need are not going to be developed in time." 

Photo 1: Objective of global priority pathogens list (global PPL).
Source
The WHO was requested by its members to develop a global priority pathogens list which will  prioritize the R&D for new antibiotics. For development of the global PPL, WHO put up a team of eight experts in infectious diseases, clinical microbiology, R&D, public health and infection control. Then, a multi-criteria decision analysis (MCDA) technique was used which allows to look into multiple alternatives, expert opinion and evidence-based data clubbed everything into one. The committee also decided to avoid organisms like Mycobacterium, HIV, malaria since they were already a priority at a global scale.

Based on the analysis, the pathogens were grouped into three priority tiers: Critical, High and Medium. The list is as follows

Priority 1: Critical
  • Acinetobacter baumannii, carbapenem-resistant 
  • Pseudomonas aeruginosa, carbapenem-resistant 
  • Enterobacteriaceae members (Klebsiella pneumonia, Escherichia coli, Enterobacter species, Serratia species, Proteus species, Providencia species, Morganella species)  that are carbapenem-resistant, 3rd generation cephalosporin-resistant. 
Priority 2: High
  • Enterococcus faecium, Vancomycin-resistant 
  • Staphylococcus aureus, Methicillin-resistant, Vancomycin intermediate and resistant 
  • Helicobacter pylori, Clarithromycin-resistant 
  • Campylobacter species, Fluoroquinolone-resistant 
  • Salmonella species, Fluoroquinolone-resistant 
  • Neisseria gonorrhoeae, 3rd generation cephalosporin-resistant, fluoroquinolone-resistant 
Priority 3: Medium
  • Streptococcus pneumoniae, penicillin-non-susceptible 
  • Haemophilus influenzae, ampicillin-resistant 
  • Shigella species, fluoroquinolone-resistant 
The panel has also noted that the above list is not the ultimate and there are a couple of limitations. For example, the decision of above overlooks that High-quality data is missing, especially for community-acquired infections and from low-income countries. The data is intended to be seen as a guideline for what is the pressing need. Mr Hermann Gröhe says, "We need effective antibiotics for our health systems. We have to take joint action today for a healthier tomorrow. Therefore, we will discuss and bring the attention of the G20 to the fight against antimicrobial resistance. WHO’s first global priority pathogen list is an important new tool to secure and guide research and development related to new antibiotics."

Reference:


GLOBAL PRIORITY LIST OF ANTIBIOTIC-RESISTANT BACTERIA TO GUIDE RESEARCH, DISCOVERY, AND DEVELOPMENT OF NEW ANTIBIOTICS. WHO publication. Link

Wednesday, March 01, 2017

BtB# 12- Why is ID a Requirement in Clinical Microbiology

I have been recently on a very tight schedule with regards to my work and hence have not been able to post for past couple of weeks. In this post, I want to talk about a very basic clinical microbiology question. A clinical microbiologist often encounters several different types of microbes in the clinical samples. For bacterial isolates, antibiotic resistance profile is tested and the treatment is decided based on the profile obtained.

So the question, why do you actually need to identify the pathogen? If you have a colony growing there, wouldn't it be sufficient to test antibiogram and then start treatment based on that? Why devote resources to go ahead and identify the pathogen? Sounds a trivial question. I have asked this question myself as an undergrad (Yeah, I know!!! The typical undergrad who keeps asking boring questions). But, if you consider that most of the times it is desirable to not only identify the species but also go ahead and identify its serotype or pathotype identity (Not in all cases), the question doesn't look so trivial.

If you can distill all the reasons, there are following essential reasons of why a clinical microbiologist like to identify the pathogen.

1. Predict the likely outcome of the infection.
2. Predict likely sensitivity to antimicrobials.
3. Obtain research information on a new disease.

There is a great depth of clinical research that has been done and thus the most likely outcomes of a given infection are almost predictable. For example, identification of E coli or Shigella dystentriae has different meanings for the expected clinical outcome when isolated from the stool sample. The treatment strategy varies accordingly and mere antibiotic sensitivity is not enough for the treating physician.

International and local data are available regarding antibiotic resistance pattern for a good number of pathogens. Certain pathogens are inertly resistant to different antibiotics. For example, Pseudomonas aeruginosa is inherently resistant to tigecycline and I will not even consider testing for tigecycline if my presumptive identification of the pathogen is P aeruginosa. The same is the case for colistin in the case of E meningoseptica. If the pathogen is known, treatment can be started more accurately even though the actual resistance profile is not known.

The third reason is that fishing can be a good research in itself. Thanks to an increasing ability conferred by genetic diagnostics, many different infections have been attributed to agents that otherwise were previously not attributed to being pathogenic. The knowledge of different species that can cause infection has significantly expanded. Interestingly, it has been the opinion that in many cases the species identification has been wrong (Shown using genetic tests) and they are in reality a different species.

The immediate next question is when is it desirable to identify upto a species level and when is it necessary to go beyond?

For most cases speciation is sufficient except for those situations where sub species has a different clinical effect. For example, it is sufficient to know for treating physician that the skin infection is caused by S aureus and is not a MRSA to treat. Knowing its clonal type and further wouldn't be of immediate patient interest, though it maybe pursued to study molecular epidemiology. For Salmonella enterica, identified from a stool sample, it is desirable to further identify it as Typhi/Paratyphi/Cholerasuis etc. It is also advisable to go further down the line, if something unusual is noticed. For example, If a particular type of resistance pattern is constantly observed from same ward it indicates a possible outbreak which should be investigated by typing.

I should end with a note. There are situations where the identification hasn't been yet done but resistance profile is available. For example, in cases of meningitis where most probably there is a single species involved, organism can be plated and on other hand a heavy inolculation can be made on MHA and antibiotics put directly. This has been shown to effective by some studies in reducing the time for reporting. In such cases, the ID is not yet available but the probable resistance profile is available to aid physician. It should be noted that this is not a confirmatory situation. This is usually followed up with routine culture, identification and sensitivity testing.

Friday, February 10, 2017

Blogger's Desk# 11- Microbiology Textbooks

In a recent blog post, I had written about my picks of microbiology blogs of interest (Link). The post gained a lot of hits. I got a few communication suggesting other blogs that were good. The post was written in response to many readers asking what blogs out there I like and read. In similar lines, a question that I have encountered from a long list of students (who are just freshers into the medical microbiology), is what textbooks are recommended? Simplest answer, there is no universally recommended book.

There is no lack of microbiology texts available in print and Ebook. Each book have their own positives and drawbacks. There are also specialised textbooks that focus elaborately on a single topic. So it is unfair for me to give a graded list of 10 books that I consider as a must read. Considering a broad range of topics, and based on my experience of reading from multiple texts, I have compiled a list. I dont endorse any publisher or company and hence the list is my independent opinion. For the same reason I will not provide any links to publisher site or other sites selling the books. You know how to find if you want one.


1. General Microbiology
Key Author- Roger Y. Stanier

The first thing that you study when starting with microbiology is understanding the general concepts. I have strongly noted, especially among my students that the basic understanding of core microbiology concepts are very weak since a lot of medical microbiology texts dont have the luxury of going through basics, especially in detail. This books has excellent chapters on bacterial classification, structure and metabolism. 

Positives: The best part of this book is the  details of explanation and the range of details of covered.
Negatives: The down part is the book hasn't been updated in ages. It has been atleast 3 decades since this book has undergone revision. If you want to understand the classic microbiology, this is the go to book.

2. Prescott's Microbiology
Key Authors: Linda Sherwood, Joanne Willey, Christopher J. Woolverton

The book is an alternative for Stanier's microbiology and introduces the reader to basic concepts of microbiology. The book is well balanced with sufficient discussion and illustration alloted to basic topics. The book is considered by many as the "Bible of basic Microbiology". 

Positives: Excllent readability and ease of language. Everything that you need to know is covered.
Negative: Nothing that I can highlight.

3. Ananthanarayan and Paniker's Textbook of Microbiology
Key Authors: R. Ananthanarayan, Ananthanarayan And Paniker

This is a classic textbook of microbiology that most students (especially south east asian students) are most familiar with. The book has basically an excellent introduction to medical microbiology concepts. Though titled as Medical Microbiology, this book covers bacteriology and virology related to medical science. The book is basically a boiled down and extracted notes of medical microbiology. The matter cannot be reduced any further.

Positives: This book is inidispensable for first time learners of medical microbiology. You get all the basics in the simplest format.
Negative: Detailed explanation is lacking for a huge number of topics. The newer editions claim that the book has been revised thourougly, but in essence newer editions are simply old wine in new bottle.

4. Principles and Practice of Infectious diseases
Key Authors: John E. Bennett, Raphael Dolin & Martin J. Blaser



No other textbook in my view comes close to this book when you want to undersatnd Infectious diseases as a whole. The book has extremely elaborate details of everything you want to know about infectious disease and is the ultimate reference book for practising infectious disease specialist. Book comes as a two volume set. The editions are frequently updated and no information is out of date. 

Positives: Depth of literature and discussion.
Negatives: The book is so extensive and elaborate and is not the one of choice for very beginers. This is a book of choice for people who have some basic understanding of medical microbiology and what to get into details.

5. Parasitic Diseases
Key authors: Dickson D. Despommier. Daniel O. Griffin. Robert W. Gwadz

The book focusses on specially Parasitic diseases. In simplest words the book is concise, accurate and student friendly. Parasitology is not well discussed in a lot of microbiology book and hence this book is illuminating. The book doesnt get into any hefty details but gives sufficient understanding  of the concepts. Another reason to read this book is, its latest edition is available online for free download legally.

Positives: Excellent explanation and fanastic readability. Illustration of life cycle are simply excellent
Negatives: Nothing that I can highlight especially since its a free to read book.


6. Antimicrobial Susceptibility Testing Protocols
Editors: Richard Schwalbe, Lynn Steele-Moore, Avery C. Goodwin

I wouldnt want to highlight a specialised textbook, focussing on a single topic in this post since such books are bound to be detailed and carefully crafted. I want to make an exception in this case, since this is the only literature that I have ever found to give a detailed explanation of antibiotic testing protocols that is easy to read, understand and execute.

Positives: Point to point explanation.
Negatives: A book that focusess on Antibiotic testing protocols should have had atleast one small chapter on antibiotic basics.

7. Fundamental Medical Mycology
Key Authors: Errol Reiss, H. Jean Shadomy, G. Marshall Lyon

Quoting from a review by Thomas G. Mitchell "This complementary team has produced a highly readable and comprehensive book, which they intend to be a text for medical and graduate students, a resource for microbiology technologists, and a reference for physicians and researchers."

Positives: Carefull organisation and clarity of discussion
Negatives: Graphical appeal of the book needs improvement.


8. Koneman's Color Atlas and Textbook of Diagnostic Microbiology
Key Authors: by Washington C. Winn, Stephen D. Allen, Stephen Allen, William M Janda, Elmer W. Koneman, Paul C. Schreckenberger, Gary W. Procop, Gail L. Woods.

Understadning microbiology concepts is one side of the story, while the second being working in a Clinical diagnostic laboratory. This book is long considered as the definitive work in its microbiology dignostics field in all aspects of clinical microbiology- Bacteriology, Mycology, Parasitology, and Virology. The book is a reference for all the diagnostic procedure in Clinical microbiology and a must read book.

Positives: The best in diagnostic microbiology procedures
Negatives: Nothing that I can highlight.

9. Mackie & Mccartney Practical Medical Microbiology
Key Author: J G Collee

A classical book in diagnotic microbiology, is a microbiology text similar to Koneman's but not as elaborate but yet discusses the subject in detail. The book forms a preliminary read if you are getting into diagnostic microbiology. Most concepts are well explained.

Positives: Easy to understand concise discussion of diagnotic workup.
Negative: The book hasn't been revised or updated since 1980's.


10. Principles of Virology
Key Authors: Jane Flint, Vincent Racaniello, Glenn Rall, Anna Marie Skalka.

Virology is a field that is quite different from other and they are radically different. The concepts governing viruses is totally different and hence a good virology book helps a lot. I relied on Fields Virology, for a long time (and still do). In contrast, this book has a different approach to talk about virology and descriptions are with reference to modern molecular understanding. The clarity and simplicty of the book is very appealing.

Positives: Simplicity and ease to understand
Negatives: Nothing that I can highlight.


Friday, February 03, 2017

CRISPR cas- Snap look

CRISPR cas system (Clustered regularly interspaced short palindromic repeats- CRISPR associated protein) is a topic that I have been writing about multiple times in this blog. Long ago I wrote a really basic post on CRISPR cas system, and since then the understanding and the technolgy has come a long way. I was recently discussing about CRISPR with a friend and though an update post would be interesting to readers.

In a previous post, I wrote about proteins that can block CRISPR. There are several arguments around this such as Phage possess anti CRISPR defense system, Phage can possess CRISPR system etc. The question becomes obvious that where did the CRISPR system develop first. Evolutionary origins can be a tough question to answer especially since there is a lot of genetic swapping between phages and bacteria and even between different bacteria. Further, there is some evidence that CRISPR genes are tough to keep and hence a lot of it is in loose and gain process. In theory, this is a chicken or egg problem for which origin cannot be confirmed with sufficient confidence.

Table 1: Classification of CRISPR-Cas systems. Source
Coming to the classification, the system is classified into 2 classes, 5 types, and 19 subtypes in all. Class 1 CRISPR–Cas systems are defined by the presence of a multisubunit crRNA–effector complex. In contrast, Class 2 contains only a single protein. Class VI is the most recent addition, the only one that can cut the RNA. Type IV is a putative system and there is significant lack of details. A brief classification is shown in Table 1. It is not clear, as how many types of CRISPR are actually there in the prokaryotic world, since there is a huge lack of sequence in bacterial population. CRISPR-Cas systems has been demonstrated in about 40% of eubacteria and 80% of archaea which is known. There is a perfect possibilty that there is something similar to CRISPR and we have not yet figured it out. Till I recently read an article by Heidi Ledford, I wasn't aware that there is some evidence that CRISPR is also important for pathogenesis at least in a few cases. There are a couple of possible explanations in the article. But what is striking is the idea that CRISPR is not just a defense system. It  has more to it. 

    Makarova K, Wolf Y, Alkhnbashi O, Costa F, Shah S, Saunders S et al. An updated evolutionary classification of CRISPR–Cas systems. Nature Reviews Microbiology. 2015;13(11):722-736. 

Savitskaya E, Musharova O, Severinov K. Diversity of CRISPR-Cas-mediated mechanisms of adaptive immunity in prokaryotes and their application in biotechnology. Biochemistry. 2016;81(7):653-661. 

Ledford H. Five big mysteries about CRISPR’s origins. Nature. 2017;541(7637):280-282.

Friday, January 27, 2017

Speaking Phage language

Host Pathogen relationship is sometimes a horribly complicated molecular network. Merely infecting a host and continuing to do so, is not the goal of the pathogen (See my earlier post). Bacteria has been well studied in this contex. Quorum sensing is a method of establishing communication with other bacteria to determine what would be the right time to carry out a particular activity. Similar communication system exist in fungal and parasitic world, though they havent been studied in extensive detail.

Viruses are different. It is not sure if we should even call the viruses as a living entity. Bacteriophages are viruses that infect bacteria with most phages having 2 types of life cycle to choose from. Though on the overview it appears that lytic or lysogenic cycle is a random choice for phage, it is not so. It is known that the number of host available to infect has an has an influence on choice of life cycle. Think about it. If there are too few bacteria for the phage to infect, most probably that whole population is going to die and progeny phage will have nothing to further proliferate. So it is more wise to be latent and wait for the bacteria to replenish in such cases. On the other way around, having a lot of bacteria to infect around would mean that it is better to go after a lytic cycle.

So how does the phage decide? The decision to undergo lytic or lysogenic cycle depends on certain molecular switches. There has been substantial enquiry into these pathways and there is a reasonable understanding of what is probably happening. However, phages by themselves communicating is not known, untill now. In a recent publication in nature, a team has for the first time, identified a peptide molecule (called as arbitrium) to communicate with one another that literally decides the life cycle choice.

The researchers started with a hypothesis that bacteria secrete communication molecules to alert other bacteria of phage infection. For this, Bacillus subtilis strain 168 were infected by four different phages- phi29, phi105, rho14 and phi3T. A chance observation led to the hypothesis that a small molecule is released to the medium during infection of B subtilis by phi3T (Phenomenon was not observed in other phages) and it probably modulates infection dynmaics of the phage. Further study showed it was a short peptide. The researchers went ahead and sequenced the phi3T genome and made came up with a couple of candidate genes. Of these, one gene exhibited features reminiscent of Bacillus quorum sensing peptides which encoded a short ORF which could be processed by B subtilis extracellular proteases (based on consensus cleavage site for peptide maturation). The predicted peptide was Ser-Ala-Ile-Arg-Gly-Ala (SAIRGA) which was subsequently confirmed by mass spec analysis. The peptide was further verified by showing that there was a dose dependent effect of  elevated lysogeny in the presence of the SAIRGA peptide. The gene was named as aimP, which is located just next to another gene that codes for tetratricopeptide repeat (TPR) domain, typical of intracellular peptide receptors of the RRNPP family in quorum sensing systems. This gene turned out to be the receptor. The rest of the paper deals with experiments to identify how this system works. 

Transcriptome analysis of phage for effect of arbitrium had a transcript (aimX), that was immediately downstream to the AimR DNA-binding site. This transcript showed substantial expression in the absence of the arbitrium but was severely reduced in presence of the peptide. Based on some more genetic knockdowns and other assays the researchers came up with a model shown in Fig 1.

Fig 1: a, Dynamics of arbitrium accumulation during infection of a bacterial culture by phage. b, At the first encounter of a phage with a bacterial population, the early genes aimR and aimP are expressed immediately upon infection. AimR, as a dimer, activates AimX expression. AimX is an inhibitor of lysogeny, possibly as a regulatory non-coding RNA, directing the phage to a lytic cycle. At the same time AimP is expressed, secreted and processed extracellularly to produce the mature peptide. c, At later stages of the infection dynamics, the arbitrium peptide accumulates in the medium and is internalized into the bacteria by the OPP transporter. Now, when the phage infects the bacterium, the expressed AimR receptor binds the arbitrium molecules and cannot activate the expression of AimX, leading to lysogeny preference. Copied from Source
Earlier, Clokie etal;2014 had published a paper indicating that bacterial quorum sensing systems can be found on phage genomes. Clokie comments on the paper, "I’ve thought about doing those experiments to see if there’s something in the media. Phages broadcast in different frequencies. They speak in different languages and they can hear only the language that they speak". Source

References

Callaway, Ewen. "Do You Speak Virus? Phages Caught Sending Chemical Messages". Nature (2017): doi:10.1038/nature.2017.21313

Erez, Zohar et al. "Communication Between Viruses Guides Lysis–Lysogeny Decisions". Nature 541.7638 (2017): 488-493. 

Hargreaves, Katherine R., Andrew M. Kropinski, and Martha R. J. Clokie. "What Does The Talking?: Quorum Sensing Signalling Genes Discovered In A Bacteriophage Genome". PLoS ONE 9.1 (2014): e85131.

Thursday, January 19, 2017

Report of a Klebsiella pneumoniae strain resistant to 26 antibiotics

Ever since MCR-1 has started making its news as an antibiotic resistance gene of interest, a lot of reports have been published around the world looking into colistin resistance. It should be noted that MCR-1 is not the only mode of colistin resistance. Here is a recent report on one such example.

In a recent publication of CDC MMWR, a Klebsiella pneumoniae isolate is described that is resistant to 26 antibiotics (This includes aminoglycosides, polymyxins, tigecycline and Colistin). The strain was also NDM positive. This case was reported from Nevada, of a woman who died in September from an incurable infection. The isolate was negative for MCR-1. Quoting from the article

The patient was a female Washoe County resident in her 70s who arrived in the United States in early August 2016 after an extended visit to India. She was admitted to the acute care hospital on August 18 with a primary diagnosis of systemic inflammatory response syndrome, likely resulting from an infected right hip seroma. The patient developed septic shock and died in early September. During the 2 years preceding this U.S. hospitalisation, the patient had multiple hospitalisations in India related to a right femur fracture and subsequent osteomyelitis of the right femur and hip; the most recent hospitalisation in India had been in June 2016.

This article raises the red flag of how serious the issue of pan-drug resistance has now become. Basically, at least in this case, there was nothing in the antibiotic panel that could have been used for treatment. Technically speaking it is a total drug resistant Klebsiella pneumoniae species. It is still called pan-drug resistance since The isolate had a relatively low fosfomycin resistance and was intermediately resistant to tigecycline.

The article mentions the possibility that this strain was acquired from India but cannot confirm the same due to several reasons. Colistin resistance in Klebsiella has been described earlier in India in 2014. However, it is absurd from the side of media to blame that colistin resistance comes from countries like India. Colistin resistance has been reported as early as 2009 in US.

Alexander Kallen, a medical officer at the CDC comments, “It was tested against everything that’s available in the United States and was not effective. I think it’s concerning. We have relied for so long on just newer and newer antibiotics. But obviously the bugs can often [develop resistance] faster than we can make new ones.”

References

A Nevada woman dies of a superbug resistant to every available antibiotic in the US. 

Chen L, Todd R, Kiehlbauch J, Walters M, Kallen A. Notes from the Field: Pan-Resistant New Delhi Metallo-Beta-Lactamase-Producing Klebsiella pneumoniae — Washoe County, Nevada, 2016. MMWR. 2017;66(1):33. 

Goel G, Hmar L, Sarkar De M, Bhattacharya S, Chandy M. Colistin-Resistant Klebsiella pneumoniae: Report of a Cluster of 24 Cases from a New Oncology Center in Eastern India. Infection Control. 2014; 35 (08):1076-1077. 

Marchaim D, Chopra T, Pogue J, Perez F, Hujer A, Rudin S et al. Outbreak of Colistin-Resistant, Carbapenem-Resistant Klebsiella pneumoniae in Metropolitan Detroit, Michigan. Antimicrobial Agents and Chemotherapy. 2010;55(2):593-599.