Category Archives: The Science of Microbiology

“When the bugs take their time…”

You are probably familiar with the scenario… A blood culture takes a couple of days to become positive. A Gram stain shows Gram negative rods. The plates are subbed but after another couple of days there is still no growth on the plates. The clinicians are getting impatient and are phoning the lab looking for an identification…

It is certainly a frustrating situation but one that occurs not infrequently.

Is there anything that can be done to help the clinician, and more importantly the patient, whilst we are waiting for the organism to grow?

There are several organisms to consider in this sort of scenario. We should be thinking about HACEK organisms, anaerobes, Pasteurella spp., oxidative non-fermentors like Burkholderia spp., Capnocytophaga spp. Consider micro-aerophilic bacteria such as Campylobacter spp. and Helicobacter spp.  And don’t forget about exotic organisms such as Brucella spp.  Even then, this list is by no means exhaustive, and I am sure there are others that you have come across that I have forgotten about!

What can the lab/clinical microbiologist do to narrow the differential down and manage accordingly pending plate growth?

A few things come to mind:

Aerobic or anaerobic bottle positive?: If the aerobic bottle only is positive it can point to non-fermentors like Burkholderia spp. If the anaerobic bottle only is positive, then one must think about anaerobes (e.g. Bacteroides spp. Fusobacterium spp. )

Gram stain appearance: A cocco-bacillary appearance should make one think of Haemophilus and Brucella. Longish, pleomorphic spindle-shaped organism on Gram point towards Capnocytophaga. “Seagulls” or “squiggles” should send you in the direction of Campylobacter/Helicobacter

Patient History: A history of dog bites/exposure should make one think of Capnocytophaga. A prosthetic valve or other valve disease, or clinical stigmata of endocarditis can indicate a HACEK organism. A history of injecting drug use makes one suspicious of Burkholderia cepacia. A travel history to an endemic area could make one think of Brucella spp. or Burkholderia pseudomallei. In a patient with neck pain and swelling, you don’t want to miss a Fusobacterium necrophorum. If the patient is frankly septic, you want to make sure they are getting covered for Capnocytophaga and Pasteurella.

Of course, all this is speculation, and our educated guesses may be completely wrong in the end. It is however important speculation… We want to make sure that the patient is being covered for the most likely and the most serious possibilities.

Taking another, but no less important angle, from a lab point of view it is essential that any slow growing Gram-negative organisms are worked up in a biohazard cabinet. Laboratory exposure incidents for organisms such as Brucella spp.  and Burkholderia pseudomallei are resource-intensive, stressful for the staff, and for the most part avoidable.

And sometimes the bugs just like to mock us, even make fools of us. Just recently, I was convinced a wavy Gram negative rod in (multiple) positive blood cultures from a patient was going to turn out to be a Campylobacter, only for it to finally be identified as a Helicobacter cinaedi… Wrong again!


“The return of the fried egg”

After a prolonged COVID-induced hibernation, Mycoplasma pneumoniae is back in NZ. Over the past couple of months there have been a sprinkling of cases throughout NZ picked up on respiratory panel testing by PCR in diagnostic laboratories, including my own.

I can hardly remember the last time I saw a Mycoplasma pneumoniae infection. It was definitely pre-COVID. The low Ro of Mycoplasma pneumoniae infection meant that transmission was brought to an almost instantaneous halt by the introduction of Non-Pharmaceutical Interventions (NPIs or “lockdowns”) following the emergence of the COVID pandemic.

Over the past year or so, it has started making a comeback worldwide. It has taken longer than other pathogens to return post-COVID, and as described in this interesting article in the Lancet, this is likely due to various factors such as the slow generation time (6 h), long incubation period (1–3 weeks), and relatively low transmission rate of Mycoplasma pneumoniae. It has returned later than most in New Zealand, likely due its geographical isolation and relatively sparse population.

In a (slightly perverted) way I am glad it is back. It is a very interesting bug! It is one of the smallest known bacteria, at only 300nm. It lacks a cell wall, which is why you cannot see it on a Gram stain. It is also why Mycoplasma infections do not respond to beta-lactam antibiotics. It most commonly causes respiratory infections but also has the ability to affect a wide range of organ systems, either by direct invasion or autoimmune reaction. This is eloquently summarised on this table.

It is sometimes referred to as a cause of “walking pneumonia” which can be a little misleading as it is certainly well capable of causing hospitalisation in both adults and children.

As for the “fried egg”, this refers to its phenotypic appearance on culture media (e.g. Hayflick media). Of course, fried eggs have not really returned as virtually nobody these days (and certainly no diagnostic labs in NZ) culture for Mycoplasma pneumoniae, with the mainstay of diagnosis now being PCR and to a much lesser extent, serological testing. Having said this, there is some evidence that Mycoplasma serology is more sensitive than PCR, likely due to delayed presentation to Health Services.

With the worldwide resurgence of Mycoplasma pneumoniae, the other thing to consider is whether the re-emerging strains are macrolide resistant.  This is difficult, due to the lack of laboratories culturing for Mycoplasma pneumoniae. However, assays are now being developed to look for the key rRNA mutations coding for macrolide resistance directly from clinical samples. It is certainly something we will need to think about both from a treatment and surveillance point of view.

Which is the optimal sample type for (molecular) diagnosis of Mycoplasma pneumoniae? There does not seem to be a lot of literature on this topic and it probably depends on the clinical presentation of the disease. If presenting as an upper respiratory tract infection, which it can do, then throat or nasopharyngeal swab is likely to be best.  For pneumonia then sputum or BAL are probably optimal.

So, Mycoplasma pneumoniae is back, and because of this, we need to start thinking about it again as a differential diagnosis in our patients. Its return has however, made my job a little more interesting…


“Playing the Odds”

I spent a lot of my medical student days working part-time in the bookmakers, where managing odds and probability underpin the industry. Mathematics was my favourite subject at school, and within that field, probability was my favourite sub-topic.

Now that I am a clinical microbiologist, I can see the similarities. Clinical microbiologists get involved in a lot of the early antimicrobial stewardship decisions for patients based on preliminary/interim microbiology results. This is an area where you definitely need to weigh up the percentages/odds.

For example if you get a positive blood culture with gram positive cocci on the Gram, what will be the probability that this will turn out to be a Staph aureus or even an MRSA? If you are awaiting susceptibilities on an E.coli bacteraemia, what will be the chances it will be an ESBL? These are decisions which have to be made when advising optimal antibiotic therapy. So many factors need to be considered, almost sub-consciously, when calculating these odds.

Of course, with all decisions that involve probability, you will never win (get it right) all of the time. This is where it is critically important to take into account the potential consequences of getting it wrong, i.e. how sick is the patient? how much “reserves” do they have, what level of care are they currently in? In a “game” which involves probabilities, it is careful and prudent management of these probabilities which is key.

The other field of probability that clinical microbiologists must have understanding of are pre- and post- test probability, and positive and negative predictive value. These are key concepts in determining the validity of any test result the laboratory produces.

Playing the odds is an integral part of being a clinical microbiologist. It may even be part of the reason I was attracted to the specialty in the first place. I can even pretend I am still working at the bookies…