Carbapenem-resistant Gram-negative bacteria are a public health threat that requires urgent action. The fact that these pathogens commonly also harbor resistance mechanisms for several other antimicrobial classes further reduces patient treatment options. The present study aimed to provide information regarding the multidrug resistance genetic background of carbapenem-resistant Gram-negative bacteria in Central Greece. Strains from a tertiary care hospital, collected during routine practice, were characterized using a DNA microarray-based assay. Various different resistance determinants for carbapenems, other beta-lactams, aminoglycosides, quinolones, trimethoprim, sulfonamides and macrolides were detected among isolates of the same sequence type. Eighteen different multidrug resistance genomic profiles were identified among the twenty-four K. pneumoniae ST258, seven different profiles among the eight K. pneumoniae ST11, four profiles among the six A. baumannii ST409 and two among the three K. oxytoca. This report describes the multidrug resistance genomic background of carbapenem-resistant Gram-negative bacteria from a tertiary care hospital in Central Greece, providing evidence of their continuous genetic evolution.

Leukocidins of Staphylococcus (S.) aureus are bicomponent toxins that form polymeric pores in host leukocyte membranes, leading to cell death and/or triggering apoptosis. Some of these toxin genes are located on prophages and are associated with specific hosts. The genes lukP/Q have been described from equine S. aureus isolates. We examined the genomes, including the lukP/Q prophages, of S. aureus strains belonging to clonal complexes CC1, CC350, CC816, and CC8115. In addition to sequencing, phages were characterised by mitomycin C induction and transmission electron microscopy (TEM). All lukP/Q prophages integrated into the lip2=geh gene, and all included also the gene scn-eq encoding an equine staphylococcal complement inhibitor. The lukP/Q prophages clustered, based on gene content and allelic variants, into three groups. One was found in CC1 and CC97 sequences; one was present mainly in CC350 but also in other lineages (CC1, CC97, CC133, CC398); and a third one was exclusively observed in CC816 and CC8115. Prophages of the latter group additionally included a rare enterotoxin A allele (sea_320E). Moreover, a prophage from a CC522 goat isolate was found to harbour lukP. Its lukF component could be regarded as chimaera comprising parts of lukQ and of lukF-P83. A putative kinase gene of 1095 basepairs was found to be associated with equine strains of S. aureus. It was also localised on prophages. However, these prophages were different from the ones that carried lukP/Q, and three different integration sites of kinase-carrying phages were identified. These observations confirmed the presence of prophage-located important virulence-associated genes in equine S. aureus and that certain prophages might determine the host specificity of the staphylococcal strains they reside in.

In the context of microarray-based epidemiological typing of the clonal organism Staphylococcus aureus/MRSA, a strain was identified that did not belong to known clonal complexes. The molecular analysis by microarray-based typing yielded signals suggesting that it was a mosaic or hybrid strain of two lineages. To verify this result, the isolate was sequenced with both, short-read Illumina and long-read Nanopore technologies and analysed in detail. This supported the hypothesis that the genome of this strain, ST6610-MRSA-IVg comprised of segments originating from two different clonal complexes (CC). While the backbone of the strain’s genome, i.e., roughly 2 megabases, belongs to CC8, a continuous insert of 894 kb (approx. 30% of the genome) originated from CC140. Beside core genomic markers in the normal succession and orientation, this insert also included the mecA gene, coding for PbP2a and causing methicillin resistance, localised on an SCCmec IVg element. This particular SCCmec type was also previously observed in CC140 MRSA from African countries. A second conspicuous observation was the presence of the trimethoprim resistance gene dfrG within on a prophage that occupied an attachment site normally used by Panton-Valentine Leucocidin phages. This observation could indicate a role of large-scale chromosomal recombination in the evolution of S. aureus as well as a role of phages in the dissemination of antibiotic resistance genes.

Staphylococcus argenteus is a recently described staphylococcal species that is related to Staphylococcus aureus but lacks the staphyloxanthin operon. It is able to acquire both resistance markers such as the SCCmec elements and mobile genetic elements carrying virulence-associated genes from S. aureus. This includes those encoding the Panton–Valentine leukocidin (PVL), which is associated mainly with severe and/or recurrent staphylococcal skin and soft tissue infections. Here, we describe the genome sequences of two PVL-positive, mecA-negative S. argenteus sequence type (ST) 2250 isolates from the United Arab Emirates in detail. The isolates were found in a dental clinic in the United Arab Emirates (UAE). Both were sequenced using Oxford Nanopore Technology (ONT). This demonstrated the presence of temperate bacteriophages in the staphylococcal genomes, including a PVL prophage. It was essentially identical to the published sequence of phiSa2wa_st78 (GenBank NC_055048), a PVL phage from an Australian S. aureus clonal complex (CC) 88 isolate. Besides the PVL prophage, one isolate carried another prophage and the second isolate carried two additional prophages, whereby the region between these two prophages was inverted. This “flipped” region comprised about 1,083,000 bp, or more than a third of the strain’s genome, and it included the PVL prophage. Prophages were induced by Mitomycin C treatment and subjected to transmission electron microscopy (TEM). This yielded, in accordance to the sequencing results, one or, respectively, two distinct populations of icosahedral phages. It also showed prolate phages which presumptively might be identified as the PVL phage. This observation highlights the significance bacteriophages have as agents of horizontal gene transfer as well as the need for monitoring emerging staphylococcal strains, especially in cosmopolitan settings such as the UAE.

Staphylococcus (S.) aureus colonizes up to 30% of all humans and can occasionally cause serious infections. It is not restricted to humans as it can also often be found in livestock and wildlife. Recent studies have shown that wildlife strains of S. aureus usually belong to other clonal complexes than human strains and that they might differ significantly with regard to the prevalence of genes encoding antimicrobial resistance properties and virulence factors. Here, we describe a strain of S. aureus isolated from a European badger (Meles meles). For molecular characterisation, DNA microarray-based technology was combined with various next-generation sequencing (NGS) methods. Bacteriophages from this isolate were induced with Mitomycin C and characterized in detail by transmission electron microscopy (TEM) and NGS. The S. aureus isolate belonged to ST425 and had a novel spa repeat sequence (t20845). It did not carry any resistance genes. The uncommon enterotoxin gene see was detected in one of its three temperate bacteriophages. It was possible to demonstrate the induction of all three prophages, although only one of them was expected to be capable of excision based on its carriage of the excisionase gene xis. All three bacteriophages belonged to the family Siphoviridae. Minor differences in size and shape of their heads were noted in TEM images. The results highlight the ability of S. aureus to colonize or infect different host species successfully, which can be attributed to a variety of virulence factors on mobile genetic elements, such as bacteriophages. As shown in the strain described herein, temperate bacteriophages not only contribute to the fitness of their staphylococcal host by transferring virulence factors, but also increase mobility among themselves by sharing genes for excision and mobilization with other prophages.

Strategies to contain the current SARS-CoV-2 pandemic rely, beside vaccinations, also on molecular and serological testing. For any kind of assay development, screening for the optimal antigen is essential. Here we describe the verification of a new protein microarray with different commercially available preparations significant antigens of SARS-CoV-2 that can be used for the evaluation of the performance of these antigens in serological assays and for antibody screening in serum samples. Antigens of other pathogens that are addressed by widely used vaccinations were also included. To evaluate the accuracy of 21 different antigens or antigen preparations on the microarray, receiver operating characteristics (ROC) curve analysis using ELISA results as reference were performed. Except for a single concentration, a diagnostic sensitivity of 1 was determined for all antigen preparations. A diagnostic specificity, as well as an area under the curve (AUC) of 1 was obtained for 16 of 21 antigen preparations. For the remaining five, the diagnostic specificity ranged from 0.942 to 0.981 and AUC from 0.974 to 0.999. The optimized assay was subsequently also applied to determine the immune status of previously tested individuals and/or to detect the immunization status after COVID-19 vaccination. Microarray evaluation of the antibody profiles of COVID-19 convalescent and post vaccination sera showed that the IgG response differed between these groups, and that the choice of the test antigen is crucial for the assay performance. Furthermore, the results showed that the immune response is highly individualized, depended on several factors (e.g., age or sex), and was not directly related to the severity of disease. The new protein microarray provides an ideal method for the parallel screening of many different antigens of vaccine-preventable diseases in a single sample and for reliable and meaningful diagnostic tests, as well as for the development of safe and specific vaccines.