Paenibacillus glucanolyticus. I don’t know how every facility doesn’t have to deal with this bug. The bug is pictured below. Any microbiologist would call this plate TNTC (too many colonies to count / >250 CFU/plate). This organism drove me crazy when I was the Plant Hygienist at a soap manufacturing plant. I referenced this event in my previous post about my recent job interview presentation. This issue also fits my series of frustrating quality problems, so I want to expand on it. Our viable air monitoring recovered P. glucanolyticus every other month near a filling line. It was always TNTC! We were required to open a tedious investigation in our quality system each time. How do you find a specific microbe source in a non-controlled warehouse staffed by hundreds of minimally gowned personnel? Let’s set the stage. Imagine a typical warehouse. 40 ft ceilings, exposed steel rafters, vents, and piping. Stamped concrete floor with painted cement brick walls. Active forklifts and whirring equipment with semi-filtered exhausts blowing air in all directions. Hundreds of employees wearing uniform pants and long sleeve shirts. Hair nets and beard covers on everyone, but nobody wore gloves unless directly working with a filler. (Side Note about this environment) There were multiple filling lines in this warehouse. They were “U” shaped like the picture below. Bottles went from a hopper onto the filling line. They were labeled, filled, then packed in boxes. At the end of the line, the boxes were palletized. Environmental monitoring didn’t have specifications, but we set action levels based on past data. Higher results signaled something wasn’t normal. The picture below represents multiple lines in the warehouse. We monitored opposing sides of the lines (the colored spots). Most recoveries were "normal" growing bacteria. We had high Paenibacillus counts in the red area. What was strange- we only got TNTC results if P. glucanolyticus was on the plate. The counts were much lower if it wasn’t there. If the organism was prevalent enough to cause TNTC results, shouldn’t it show up at counts between 1 and 250 sometimes? I found interesting research about the organism. When growing on media, individual cells can migrate away from a starting colony and form their own colonies. When the plate is read 5 days later, the colonies will look completely distinct, like the picture at the top of this post. When multiple cells do this, and multiple cells from those colonies repeat the process, your single CFU at the onset looks like hundreds of colonies after incubation. I’ve since found a time-lapse video that shows this effect. A research team at Cornell created it. They placed a few CFUs in the small circle at the top of the plate. You can see how the organism grows and what the plate looks like at the end. Definitely not the traditional growth pattern with all generations contained in the same colony. I really wish this video existed when I struggled with this organism: Knowing this- knowing your TNTC result was probably representative of only a few CFUs- what do you do next? Can you just say the TNTC result isn’t real and stop the investigation? Unfortunately, we didn’t have time-lapse footage of our plates, so we couldn’t ignore the results. Any individual plate could have been caused by TNTC CFUs. We even had this organism show up as TNTC on a product plate, so we had to discard a batch due to it. This info about the organism made my investigation harder. I couldn’t just look for obvious signs of mass contamination in the area. I now had to find a source in this giant warehouse where the organism was merely surviving. Luckily, a week of expanded EM around the line pin-pointed the source. We collected air samples from multiple locations around the line. We sampled each of those spots every shift for 7 days, averaged the results (setting TNTC to 250), and made a heat map like this: The bottle unscrambler was in the hottest spot. This chaotic machine somehow transferred bottles from the hopper to the filling line by spinning them around really fast. A maintenance mechanic helped me take it apart and douse it in sporicide. I was told maintenance cleans the unscrambler during the yearly PM, but the presence of a type of bottle that hadn’t been used in 3 years in some of the crevices determined that was a lie.
I worked at this company for another year after cleaning the unscrambler. We didn’t see the organism again during that year, so I’m confident this cleaning (plus the PM cleaning instruction update) was effective. Of course, this organism popped up as TNTC again at the next company I worked for. How have I never heard other Micro labs struggling with it?
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