Multidrug-resistant Enterobacterales (MDRE) are an emerging threat to global health, leading to rising health care costs, morbidity and mortality. Multidrug-resistance is commonly caused by different β-lactamases (e.g., ESBLs and carbapenemases), sometimes in combination with other resistance mechanisms (e.g., porin loss, efflux). The continuous spread of MDRE among patients in hospital settings and the healthy population require adjustments in healthcare management and routine diagnostics. Rapid and reliable detection of MDRE infections as well as gastrointestinal colonization is key to guide therapy and infection control measures. However, proper implementation of these strategies requires diagnostic methods with short time-to-result, high sensitivity and specificity. Therefore, research on new techniques and improvement of already established protocols is inevitable. In this review, current methods for detection of MDRE are summarized with focus on culture based and molecular techniques, which are useful for the clinical microbiology laboratory.

Intracellular Salmonella enterica serovar Typhimurium (STM) deploy the Salmonella Pathogenicity Island 2-encoded type III secretion system (SPI2-T3SS) for the massive remodeling of the endosomal system for host cells. This activity results in formation of an extensive interconnected tubular network of Salmonella-induced filaments (SIFs) connected to the Salmonella-containing vacuole (SCV). Such network is absent in cells infected with SPI2-T3SS-deficient mutant strains such as ΔssaV. A tubular network with reduced dimensions is formed if SPI2-T3SS effector protein SseF is absent. Previous single cell live microscopy-based analyses revealed that intracellular proliferation of STM is directly correlated to the ability to transform the host cell endosomal system into a complex tubular network. This network may also abrogate host defense mechanisms such as delivery of antimicrobial effectors to the SCV. To test the role of SIFs in STM patho-metabolism, we performed quantitative comparative proteomics of STM recovered from infected murine macrophages. We infected RAW264.7 cells with STM wild type (WT), ΔsseF or ΔssaV strains, recovered bacteria 12 h after infection and determined proteome compositions. Increased numbers of proteins characteristic for nutritional starvation were detected in STM ΔsseF and ΔssaV compared to WT. In addition, STM ΔssaV, but not ΔsseF showed signatures of increased exposure to stress by antimicrobial defenses, in particular reactive oxygen species, of the host cells. The proteomics analyses presented here support and extend the role of SIFs for intracellular lifestyle of STM. We conclude that efficient manipulation of the host cell endosomal system by effector proteins of the SPI2-T3SS contributes to nutrition, as well as to resistance against antimicrobial host defense mechanisms. The facultative intracellular bacterium Salmonella enterica has evolved sophisticated mechanisms to adapt to life inside a pathogen-containing vacuole in mammalian host cells. Intracellular Salmonella manipulate the host cell endosomal system resulting in formation of a complex network of tubular vesicles, termed Salmonella-induced filaments (SIFs). We applied quantitative proteomics to intracellular Salmonella in murine macrophages and compared the wild-type strain to mutant strains with aberrant SIF architecture, or no capacity for induction of SIF. We determined that those mutant strains contain higher amounts of transporters for nutrient uptake, and lower amounts of proteins for central carbon metabolism. These observations indicate response to nutrient restriction in absence of fully established SIF. In addition, the mutant strain unable to induce SIF formation showed increased amounts of proteins required for response to antimicrobial factors of the host cells. These data show that the massive remodeling of the endosomal system of host cells by intracellular Salmonella serves to essential needs, i.e. to enable access to nutrients for efficient proliferation of the pathogen, and to withstand hostile conditions within the pathogen-containing vacuole.

The facultative intracellular pathogen Salmonella enterica resides in a specific membrane-bound compartment termed Salmonella-containing vacuole (SCV). Despite being segregated from access to metabolites in the host cell cytosol, Salmonella is able to efficiently proliferate within the SCV. We set out to unravel the nutritional supply of Salmonella in the SCV with focus on amino acids. We studied the availability of amino acids by generation of auxotrophic strains for alanine, asparagine, aspartate, glutamine and proline in a macrophage (RAW264.7) and an epithelial (HeLa) cell line and examined the access to extracellular nutrients for nutrition. Auxotrophies for alanine, asparagine or proline attenuated intracellular replication in HeLa cells, while aspartate, asparagine or proline auxotrophies attenuated intracellular replication in RAW264.7 macrophages. The different patterns of intracellular attenuation of alanine or aspartate auxotrophic strains supports distinct nutritional conditions in HeLa cells and RAW264.7 macrophages. Supplementation of medium with individual amino acids restored the intracellular replication of mutant strains auxotrophic for asparagine, proline or glutamine. Similarly, a mutant strain deficient in succinate dehydrogenase was complemented by extracellular addition of succinate. Complementation of intracellular replication of auxotrophic Salmonella by external amino acids was possible if bacteria were proficient in induction of Salmonella-induced filaments (SIF), but failed for a SIF-deficient background. We propose that the ability of intracellular Salmonella to redirect host cell vesicular transport provides access of amino acids to auxotrophic strains, and more general, is essential to continuously supply bacteria within the SCV with nutrients.

Salmonella enterica serovar Typhimurium (STM) is exposed to reactive oxygen species (ROS) originating from aerobic respiration, antibiotic treatment, and the oxidative burst occurring inside the Salmonella-containing vacuole (SCV) within host cells. ROS damage cellular compounds, thereby impairing bacterial viability and inducing cell death. Proteins containing iron–sulfur (Fe–S) clusters are particularly sensitive and become non-functional upon oxidation. Comprising five enzymes with Fe–S clusters, the TCA cycle is a pathway most sensitive toward ROS. To test the impact of ROS-mediated metabolic perturbations on bacterial physiology, we analyzed the proteomic and metabolic profile of STM deficient in both cytosolic superoxide dismutases (ΔsodAB). Incapable of detoxifying superoxide anions (SOA), endogenously generated SOA accumulate during growth. ΔsodAB showed reduced abundance of aconitases, leading to a metabolic profile similar to that of an aconitase-deficient strain (ΔacnAB). Furthermore, we determined a decreased expression of acnA in STM ΔsodAB. While intracellular proliferation in RAW264.7 macrophages and survival of methyl viologen treatment were not reduced for STM ΔacnAB, proteomic profiling revealed enhanced stress response. We conclude that ROS-mediated reduced expression and damage of aconitase does not impair bacterial viability or virulence, but might increase ROS amounts in STM, which reinforces the bactericidal effects of antibiotic treatment and immune responses of the host.

Magnusiomyces clavatus and Magnusiomyces capitatus are emerging yeasts with intrinsic resistance to many commonly used antifungal agents. Identification is difficult, and determination of susceptibility patterns with commercial and reference methods is equally challenging. For this reason, few data on invasive infections by Magnusiomyces spp. are available.