Microplastic associated Enterobacteriaceae biofilms in marine sewage plant outfalls

Abstract

Microplastics are small (<5 mm) plastic debris and cause a virtually ubiquitous form of pollution in marine settings. They have been found in all compartments ranging from sea ice to sediments, beaches, and the open ocean. Microplastics are known to adsorb chemical pollutants but also provide a surface for biofilm formation by microorganisms. They originate from land-based sources including wastewater treatment outfalls. Policy, at least in the South African context, dictates that partially treated wastewater from coastal cities could be deposited into the ocean. Plastics, and microplastics in particular, represent a considerable proportion of this mixture and includes a consortium of microorganisms, specifically Enterobacteriaceae. These bacteria are ubiquitously associated with gut content of humans and other animals. Furthermore, they may be resistant to antibiotics. Such antibiotic resistant Enterobacteriaceae, may form biofilms on the microplastics and these could be dispersed into oceans. This study aimed to isolate Enterobacteriaceae species from virgin and environmental microplastics that originated from a wastewater treatment plant (WWTP) dispersing into the ocean through a marine sewage outfall. This was achieved by utilizing a simulation. Microcosms were set up by spiking seawater and artificial seawater with WWTP effluent and adding the plastic pieces. Scanning electron microscopy (SEM) was used to determine colonization on the microplastics. Selective media and incubation conditions were used to isolate Enterobacteriaceae. Pure isolates were tested against 16 antibiotics normally used in a clinical environment. Dominant species identified were Citrobacter sp., Escherichia sp., Enterobacter sp., Raoultella sp., Klebsiella sp. as well as Aeromonas sp., and Pseudomonas sp.. It is disquieting that some of these species were isolated from all three compartments of the wastewater treatment train, potentially demonstrating their extreme ability to survive, most probably due to protection in the biofilm. Resistance to beta-lactam antibiotics (ampicillin and augmentin – ampicillin-cluvanate) was common and ranged from 68% to 89% amongst isolates. Furthermore, some of these were also resistant to carbapenems (doripenem and imipenem; 9% to 27%) or, in some cases, showed reduced susceptibility to these (27% to 32%). Some of the isolates were resistant to up to ten of the antibiotics tested. Based on their ability to produce extracellular enzymes, a considerable number of these isolates could be classified as potential pathogens. PCR analysis showed that many of the isolates contained the genes intl1, blaFOX and blaMOX. Bacteria containing the intl1 gene have the ability to transfer the carbapenem and also other antibiotic resistance genes to related and non-related species in biofilms, where they enjoy extreme close proximity to each other. Enterobacteriaceae can cause a wide variety of illnesses such as gastroenteritis and urinary tract infections. Bacterial infections caused by Carbapenem-resistant Enterobacteriaceae that harbour multidrug resistance has become a global concern in the fight against bacterial infections. The results of the present project show that clinically relevant Enterobacteriaceae colonize microplastics and that these survive in biofilms on these microplastics surfaces. The concern is that the colonisers could be pathogens or opportunistic pathogens and could disperse into oceans causing a global threat to other organisms using this habitat. In addition, the isolates may have or acquire antibiotic resistance (horizontal gene transfer though the intl1 gene) in such biofilm, exacerbating the spread of the pathogens as well as antibiotic resistance traits. Such data suggest that studies should be undertaken that will challenge the current policy regulating the release of sewage through marine outfalls as well as the notion that dilution of sewage is an answer to pollution. Given the global rise in antibiotic resistant infection and annual mortality expected to increase to 10 million by 2050, finding ways to reduce antibiotic-resistant bacteria and the genes into the environment is important. In the case of the present project, this will bring into focus reducing the amount microplastics that enter the oceans, which may address two of the current looming environmental catastrophes, namely pollutant microplastics and antibiotic-resistance.