Among the leading causes of bacteremia are Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. E. coli and K. pneumoniae are increasingly exhibiting resistance to last-resort antibiotics, such as carbapenems. Rapid and accurate identification of these pathogens is critical for timely treatment and infection control. This paper aimed to develop a computer-aided bacterial morphometric technique for identifying and classifying wild-type E. coli, K. pneumoniae, and S. aureus in a field guide fashion. A 3D laser scanning confocal microscope was used to gather key parameters of each organism: length (L, µm), circular diameter (CD, µm), volume (V, µm³), surface area-to-cross-sectional area ratio (SA/CSA, unitless), surface uniformity ratio (Str), and surface texture ratio (Sdr). Microscope images and measurement results showed that S. aureus was spherical with the shortest length (1.08 µm) and smallest volume (0.52 µm³). E. coli and K. pneumoniae were rod-shaped with lengths >2.0 µm and volumes >1.0 µm³. Carbapenem-resistant (CR) strains exhibited larger volumes than their wild-type counterparts. Surface parameters further differentiated strains: wild-type E. coli had a greater surface texture or a less smooth surface (larger Sdr) than K. pneumoniae (lower Sdr) did. CR E. coli had more surface uniformity (lower Str) than CR K. pneumoniae did. A dichotomous key based on shape, circular diameter, volume, length, and surface characteristics was developed to classify the species using a series of paired, contrasting features. This morphometric analysis can aid researchers in quickly identifying bacteria, leading to faster diagnosis of life-threatening diseases and improved treatment decisions.

This paper aims to present the phenotypic characteristics, such as length, width, circular diameter, volume, surface area to cross-sectional area ratio, surface uniformity, and surface texture, of the foodborne pathogens Salmonella enterica serovar Agona, Salmonella enterica serovar Enteritidis, Listeria monocytogenes, and Campylobacter jejuni. It is a novel “field-guide” presentation of the observable morphological characteristics of these four species in the form of a Dichotomous Key. Phenotypic values of the pathogens were measured using a high-accuracy, nano-level-resolution 3D laser confocal scanning microscope. All samples were fixed with glutaraldehyde and stained with methylene blue, ensuring morphological preservation. The images of the pathogens were captured under various imaging modes, including 3D, laser, and transmission electron microscopy resolutions. Results show that these pathogens exhibit distinct morphological and surface properties, with Campylobacter jejuni showing unique features. The dichotomous key shows a simplified way of classifying selected foodborne pathogens from their morphometric properties. The morphometric measurements, in many instances first reported in the literature, and images provide a readily observable way to identify and classify microorganisms, allowing researchers to potentially study evolutionary relationships, assess species diversity, and understand how organisms interact with their environment, especially when genetic information is limited or difficult to obtain.