Aims The study aimed to explore the diversity of culturable microbiota colonizing the alimentary tract and outer surfaces of German cockroaches (Blattella germanica) captured in a health care facility. Methods and Results Microbial identification was conducted using Matrix Assisted Laser Desorption–Ionization Time‐of‐Flight Mass Spectrometry (MALDI‐TOF MS) Biotyper and 16S rRNA sequencing. A total of 181 bacteria strains were isolated from 25 cockroach specimens and the MALDI‐TOF MS‐based assay yielded direct identification of 96·5% (175 out of 181) of the strains at the species level. The proteomic fingerprinting mainly revealed strains belonged to Gram‐negative Enterobacteria (103) with six different genera that were characterized including Citrobacter, Klebsiella, Kluyevera, Leclercia, Morganella and Serratia. In addition, Pseudomonas sp. strains ranked in second with 29·8% (54 strains) followed by Staphylococcus sp. (6·62%) and Enterococcus sp. (1·65%). A large number of these bacteria (n = 90, 49·72%) was found in cockroaches captured in the maternity ward, whereas 45 strains (24·8%) were recovered in the paediatric ward. Altogether, 24 bacterial species were identified from both the external surface and digestive tract of the insect, of which Serratia marcescens presented the major group (n = 80, 44·19%) followed by Pseudomonas aeruginosa (n = 53, 29·28%) and Klebsiella oxytoca (n = 9, 4·94%). Conclusion The findings showed a high prevalence of bacterial pathogens harboured in the body and alimentary tract of B. germanica captured in a health care facility. Significance and Impact of the Study This investigation shows the possible role of German cockroaches as a source for bacterial pathogens, increasing the likelihood of direct transmission of healthcare associated infections, and thereby representing a public health risk. In addition, the present study revealed a high discriminatory power of the mass spectra investigation and a competent bacterial typing tool that extends phenotypic and genotypic approaches, which allows new possibilities for fast and accurate diagnosis in medical entomology.

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