Detection of multidrug-resistant bacteria in the occupied Palestinian territory: a cross-sectional study

Background Antimicrobial resistance is a worldwide threat to public health. WHO has created several resolutions and strategies on this subject at the World Health Assembly. In May, 2015, WHO published a global action plan to mitigate antimicrobial resistance, including tracking and global surveillance focusing on improving awareness and understanding of this issue. The aim of this study was to screen for carbapenem-resistant bacteria in the occupied Palestinian Territory, to investigate the mechanisms behind the resistance, and to assess the scope of this difficulty in the area. Methods During 6 weeks in 2012, we collected all available Gram-negative isolates taken from inpatients and outpatients in hospital laboratories at Al-Shifa Hospital and five additional hospitals in the West Bank to screen for carbapenem resistance. Resistant isolates were identified with MALDI-TOF, mapped for their resistance pattern, and

Comprehensive Identification of Protein Substrates of the Dot/Icm Type IV Transporter of Legionella pneumophila

A large number of proteins transferred by the Legionella pneumophila Dot/Icm system have been identified by various strategies. With no exceptions, these strategies are based on one or more characteristics associated with the tested proteins. Given the high level of diversity exhibited by the identified proteins, it is possible that some substrates have been missed in these screenings. In this study, we took a systematic method to survey the L. pneumophila genome by testing hypothetical orfs larger than 300 base pairs for Dot/Icm-dependent translocation. 798 of the 832 analyzed orfs were successfully fused to the carboxyl end of β-lactamase. The transfer of the fusions into mammalian cells was determined using the β-lactamase reporter substrate CCF4-AM. These efforts led to the identification of 164 proteins positive in translocation. Among these, 70 proteins are novel substrates of the Dot/Icm system. These results brought the total number of experimentally confirmed Dot/Icm substrates to 275. Sequence analysis of the C-termini of these identified proteins revealed that Lpg2844, which contains few features known to be important for Dot/Icm-dependent protein transfer can be translocated at a high efficiency. Thus, our efforts have identified a large number of novel substrates of the Dot/Icm system and have revealed the diverse features recognizable by this protein transporter

Chemical Genetics Reveals Bacterial and Host Cell Functions Critical for Type IV Effector Translocation by Legionella pneumophila

Delivery of effector proteins is a process widely used by bacterial pathogens to subvert host cell functions and cause disease. Effector delivery is achieved by elaborate injection devices and can often be triggered by environmental stimuli. However, effector export by the L. pneumophila Icm/Dot Type IVB secretion system cannot be detected until the bacterium encounters a target host cell. We used chemical genetics, a perturbation strategy that utilizes small molecule inhibitors, to determine the mechanisms critical for L. pneumophila Icm/Dot activity. From a collection of more than 2,500 annotated molecules we identified specific inhibitors of effector translocation. We found that L. pneumophila effector translocation in macrophages requires host cell factors known to be involved in phagocytosis such as phosphoinositide 3-kinases, actin and tubulin. Moreover, we found that L. pneumophila phagocytosis and effector translocation also specifically require the receptor protein tyrosine phosphate phosphatases CD45 and CD148. We further show that phagocytosis is required to trigger effector delivery unless intimate contact between the bacteria and the host is artificially generated. In addition, real-time analysis of effector translocation suggests that effector export is rate-limited by phagocytosis. We propose a model in which L. pneumophila utilizes phagocytosis to initiate an intimate contact event required for the translocation of pre-synthesized effector molecules. We discuss the need for host cell participation in the initial step of the infection and its implications in the L. pneumophila lifestyle. Chemical genetic screening provides a novel approach to probe the host cell functions and factors involved in host–pathogen interactions

Phagocytosis of Staphylococcus aureus by Macrophages Exerts Cytoprotective Effects Manifested by the Upregulation of Antiapoptotic Factors

It is becoming increasingly apparent that Staphylococcus aureus are able to survive engulfment by macrophages, and that the intracellular environment of these host cells, which is essential to innate host defenses against invading microorganisms, may in fact provide a refuge for staphylococcal survival and dissemination. Based on this, we postulated that S. aureus might induce cytoprotective mechanisms by changing gene expression profiles inside macrophages similar to obligate intracellular pathogens, such as Mycobacterium tuberculosis. To validate our hypothesis we first ascertained whether S. aureus infection could affect programmed cell death in human (hMDMs) and mouse (RAW 264.7) macrophages and, specifically, protect these cells against apoptosis. Our findings indicate that S. aureus-infected macrophages are more resistant to staurosporine-induced cell death than control cells, an effect partly mediated via the inhibition of cytochrome c release from mitochondria. Furthermore, transcriptome analysis of human monocyte-derived macrophages during S. aureus infection revealed a significant increase in the expression of antiapoptotic genes. This was confirmed by quantitative RT-PCR analysis of selected genes involved in mitochondria-dependent cell death, clearly showing overexpression of BCL2 and MCL1. Cumulatively, the results of our experiments argue that S. aureus is able to induce a cytoprotective effect in macrophages derived from different mammal species, which can prevent host cell elimination, and thus allow intracellular bacterial survival. Ultimately, it is our contention that this process may contribute to the systemic dissemination of S. aureus infection

Inhibition of Host Vacuolar H+-ATPase Activity by a Legionella pneumophila Effector

Legionella pneumophila is an intracellular pathogen responsible for Legionnaires' disease. This bacterium uses the Dot/Icm type IV secretion system to inject a large number of bacterial proteins into host cells to facilitate the biogenesis of a phagosome permissive for its intracellular growth. Like many highly adapted intravacuolar pathogens, L. pneumophila is able to maintain a neutral pH in the lumen of its phagosome, particularly in the early phase of infection. However, in all cases, the molecular mechanisms underlying this observation remain unknown. In this report, we describe the identification and characterization of a Legionella protein termed SidK that specifically targets host v-ATPase, the multi-subunit machinery primarily responsible for organelle acidification in eukaryotic cells. Our results indicate that after being injected into infected cells by the Dot/Icm secretion system, SidK interacts with VatA, a key component of the proton pump. Such binding leads to the inhibition of ATP hydrolysis and proton translocation. When delivered into macrophages, SidK inhibits vacuole acidification and impairs the ability of the cells to digest non-pathogenic E. coli. We also show that a domain located in the N-terminal portion of SidK is responsible for its interactions with VatA. Furthermore, expression of sidK is highly induced when bacteria begin to enter new growth cycle, correlating well with the potential temporal requirement of its activity during infection. Our results indicate that direct targeting of v-ATPase by secreted proteins constitutes a virulence strategy for L. pneumophila, a vacuolar pathogen of macrophages and amoebae

Detection of NDM-2-producing Acinetobacter baumannii and VIM-producing Pseudomonas aeruginosa in Palestine

The aim of this study was to screen for carbapenem-resistant Gram-negative bacteria in Palestine and subsequently to identify and investigate the mechanisms of resistance. For a period of 6 weeks, all Gram-negative isolates were collected from six Palestinian hospital laboratories and were tested for susceptibility using 10 μg meropenem disks. Isolates showing resistance to meropenem were further investigated. The presence of carbapenemases was assessed by PCR. In addition, antimicrobial susceptibility testing, an efflux pump inhibitor assay and pulsed-field gel electrophoresis (PFGE) were performed. Isolates producing carbapenemases were further investigated by multilocus sequence typing (MLST). In total, 248 Gram-negative isolates were collected from the six laboratories. Among the 248 tested isolates, 15 Acinetobacter baumannii and 6 Pseudomonas aeruginosa were resistant to meropenem. One A

A Legionella pneumophila-translocated substrate that is required for growth within macrophages and protection from host cell death

Legionella pneumophila requires the Dot/Icm protein translocation system to replicate within host cells as a critical component of Legionnaire's pneumonia. None of the known individual substrates of the translocator have been shown to be essential for intracellular replication. We demonstrate here that mutants lacking the Dot/Icm substrate SdhA were severely impaired for intracellular growth within mouse bone marrow macrophages, with the defect absolute in triple mutants lacking sdhA and its two paralogs. The defect caused by the absence of the sdhA family was less severe during growth within Dictyostelium discoideum amoebae, indicating that the requirement for SdhA shows cell-type specificity. Macrophages harboring the L. pneumophila sdhA mutant showed increased nuclear degradation, mitochondrial disruption, membrane permeability, and caspase activation, indicating a role for SdhA in preventing host cell death. Defective intracellular growth of the sdhA(−) mutant could be partially suppressed by the action of caspase inhibitors, but caspase-independent cell death pathways eventually aborted replication of the mutant

The protein SdhA maintains the integrity of the Legionella-containing vacuole

Legionella pneumophila directs the formation of a specialized vacuole within host cells, dependent on protein substrates of the Icm/Dot translocation system. Survival of the host cell is essential for intracellular replication of L. pneumophila. Strains lacking the translocated substrate SdhA are defective for intracellular replication and activate host cell death pathways in primary macrophages. To understand how SdhA promotes evasion of death pathways, we performed a mutant hunt to identify bacterial suppressors of the ΔsdhA growth defect. We identified the secreted phospholipase PlaA as key to activation of death pathways by the ΔsdhA strain. Based on homology between PlaA and SseJ, a Salmonella protein associated with vacuole degradation, we determined the roles of SdhA and PlaA in controlling vacuole integrity. In the absence of sdhA, the Legionella-containing vacuole was unstable, resulting in access to the host cytosol. Both vacuole disruption and host cell death were largely dependent on PlaA. Consistent with these observations, the ΔsdhA strain colocalized with galectin-3, a marker of vacuole rupture, in a PlaA-dependent process. Access of ΔsdhA strains to the macrophage cytosol triggered multiple responses in the host cell, including degradation of bacteria, induction of the type I IFN response, and activation of inflammasomes. Therefore, we have demonstrated that the Legionella-containing vacuole is actively stabilized by the SdhA protein during intracellular replication. This vacuolar niche affords the bacterium protection from cytosolic host factors that degrade bacteria and initiate immune responses