ANTIMICROBIAL RESISTANCE

A POST ON ANTIMICROBIAL RESISTANCE

Global microbiology arrived in our small laboratory last week with the isolation of a Klebsiella pneumoniae, that appeared to be resistant to all antibiotics. It was from the sputum of a post operative patient, who had been in the hospital for two weeks.

Below is a photo of the disc sensitivity plate: 

What does this finding mean for a small rural hospital laboratory, and how did we deal with it ?

To begin with, it may be  useful to have an update on how increasing antibiotic resistance in Gram negative bacteria such as E.coli and Klebsiella spp has evolved over the last 10 years, and to understand some of the terms such as ESBL, Carbapenemase producers, metallobeta-lactamases,  VIM and NDM-1, terms you may have seen if you have seen in recent microbiology/infection journals.

Some readers of the post will know all of this, but there may be some who have not had the opportunity to keep up to date. So this discussion is mostly for the latter readers.

You will remember that beta lactamases are enzymes produced by many bacteria, that break down the structure of penicillin and similar antibiotics, so that the antibiotic can no longer kill the bacteria. Extended spectrum beta lactamases (ESBL's) are, as it says, beta lactamases with a spectrum of activity against a wider range of penicillin and cephalosporin antibiotics, so limiting the treatment options for infections caused by ESBL producing bacteria.

In the disc sensitivity testing, an E.coli or Klebsiella that has no zone to a third generation cephalosporin (cefotaxime, ceftazidime, cefixime etc), should be considered as an ESBL producer. A further test is required to determine if it definitely is an ESBL producer, one commonly used is the double disc test:

Method:

The double disc method compares the zone sizes of the isolate around a cefotaxime disc, and a combined cefotaxime plus clavulanic acid disc. A greater than 5mm diference between the zone size around the combined disc (the larger zone) and the cefotaxime disc confirms ESBL production.

The slide below shows a positive ESBL result.

While some rural hospital laboratories may be able to set this up, it may be better that the regional hospital has this facility, and smaller laboratories send suspected ESBL isolates to the regional laboratory.

 When considering antimicrobial resistance,  two components need to be considered:

a) the mechanism of resistance, eg a drug destroying enzyme such as a beta lactamase, the blocking of antibiotic uptake (tetracycline resistance), producing an alternative metabolic pathway (trimethoprim resistance)

b) the bacterial genes that code for the resistance mechanism, and how they are acquired by a bacteria.

One of the primary reasons why antibiotic resistance spreads both locally and regionally is that in many cases the genes coding for a resistance mechanism can be transferred between bacteria, and often a group of genes, coding for several resistance mechanisms, can be transferred together.

This is why we may see an E.coli that is an ESBL producer, and therefore resistant to penicillins and cephalosporins, and also resistant to aminoglycosides (gentamicin, amikacin), and to fluoroquinolones such as ciprofloxacin.

If such an E.coli was isolated from a blood culture, or a urinary tract infection, the options for treatment are very limited, and this is what is increasingly happening, even in rural tropical areas.  In many places, there would be no further antibiotic available, making treatment impossible.

The main group of antibiotics that can be used to treat such infections are the carbapenems, of which imipenem and meropenem are the main examples. Where these drugs are available, particularly in hospitals, they have been used to treat these highly resistant Gram negative infections.

However, a few years back, E.coli and Klebsiella strains were isolated that had also become resistant to carbapenems in addition to the other antibiotics. Investigations showed that they produced an enzyme, similar to beta lactamase called a carbapenemase, which inactivated the carbapenem antibiotic.

Below is a sensitivity plate with a Klebsiella pneumoniae resistant to imipenem and meropenem:

These new beta lactamases were found to have a metal ion in their molecule, and were called metallo-beta-lactamases. One of the first ones isolated and investigated was in New Delhi, India, and was labelled as New Delhi metallo-beta-lactamase-1, or NDM-1, a term you may have seen. Other metallo-beta-lactamases have been described, with names including VIM and OXA-48.

As with other resistance mechanisms, these are coded for by specific genes, which can move between bacteria. Therefore, an E.coli, which is an ESBL producer and resistant also to gentamicin and ciprofloxacin, could acquire the gene for NDM-1, and be resistant also to meropenem.

As with ESBL's, while the initial disc testing with resistance to meropenem/imipenem suggests a probable carbapenemase producer, further testing is required to confirm this. A special disc test (the Hodge test) can be used, but this is difficult to set up and monitor, and would more accurately be done at a regional laboratory. Specialised laboratories now use molecular tests (which we will talk about in post 11) to detect specific carbapenemase genes such as NDM-1.

Well, that's the end of the lecture ! 

So back to the question, if you start isolating resistant bacteria, whether ESBL producers or worse, what should the laboratory, and the hospital do about it?

Here is our experience.

The isolation of this multi-resistant Klebsiella made us realise that we didn't actually know how many times in the past we had isolated bacteria with resistance to most of our commonly available antibiotics, even if not as multi-resistant as this one. While we kept a day book of which bacteria had been isolated from which specimen (and date, patient details, etc) we had not been recording the sensitivity profile.

So, this was the first action. To begin a new day book (either an actual register, or on a spreadsheet on the computer), with columns in which to put sensitivity or resistance to the antibiotics tested. We in fact decided to have both a day book register, which the lab staff are used to using, but, as we had a computer with Excel on it, also to set up a spreadsheet. The spreadsheet  is updated from the day book at the end of each day. The advantage of the spreadsheet is that each month we can easily analyse how many isolates have been resistant to different antibiotics, how many are multi resistant. Also we can produce graphs and charts that we can use in a presentation to the doctors for them to be aware of the problem of antibiotic resistance. More details of setting up spreadsheets and databases to monitor infections will be given in post 9.

The second action was to be sure that our sensitivity testing was in fact accurate. We use the CSLI disc diffusion method. From a survey that was done in laboratories in district hospitals in our region last year, including sending round some test samples for comparison, it was evident that different laboratories were getting different results for the same isolate , and not all were following the correct criteria for the CLSI method that they said they were using.

One of the main problems was that rather than accurately measuring the zone diameter of "no growth" around a disc and comparing this to the standard table, laboratory staff were just comparing by eye zone sizes, writing down a "large" zone as "sensitive" and a small zone sometimes as "resistant" and sometimes as "partially sensitive".

This is illustrated in the image below:

We decided to look carefully at the CLSI protocol (ref link ) to ensure we were doing sensitivity testing correctly. You can check the link to assess your methods. The most important points to follow we realised are:

• Have some control organisms from the regional laboratory (E.coli, S.aureus, Pseudomonas aeruginosa), and do disc sensitivity with these and check against the CLSI control table that the zone sizes for different antibiotic discs with these control organisms are correct. This ensures that our stock of discs are potent, and our culture conditions are correct
• When testing clinical isolates, compare the zone sizes with the CLSI clinical samples table, and decide whether isolates are sensitive or resistant based on these sizes, not just by looking at the zone size.

A detailed description of the CLSI method is available at the following website:

http://www.microbelibrary.org/component/resource/laboratory-test/3189-kirby-bauer-disk-diffusion-susceptibility-test-protocol

An example of cut-off zone sizes for a range of antibiotics is given in the table below:

  This has been a rather long post! but it is an important topic, we have had to learn new things, and we hope it has been useful to you.

Further reading on Antimicrobial Resistance:

1. Increased multi-drug resistant E.coli from hospitals in Khartoum State, Sudan. Afr Health Sci 2012; 12:368-375.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3557680/

2. WHO global report on antimicrobial resistance and surveillance.
http://www.who.int/drugresistance/documents/surveillancereport/en/