Antimicrobial susceptibility in community-acquired bacterial pneumonia in adults.

OBJECTIVES: To determine the antimicrobial susceptibility patterns of Streptococcus pneumoniae and Haemophilus influenzae, two bacterial pathogens commonly associated with community-acquired pneumonia. DESIGN: Cross-sectional study. SETTING: Bacterial isolates were obtained from adults suspected to have community-acquired pneumonia and who sought treatment at two city council clinics in Nairobi, Kenya. Susceptibility to antimicrobial agents was performed using a microdilution broth method, according to the criteria set by the National Committee for Clinical Laboratory Standards. RESULTS: A total of 277 S. pneumoniae and 58 H. influenzae were obtained from 536 adults examined in the period January 1998 to December 1999. Of the 277 S. pneumoniae, only 56.7% were susceptible to penicillin and 7.6% of strains were resistant to two or more antimicrobial agents. Of the 58 H. influenzae strains, 91.4% were sensitive to ampicillin, with 6.8% resistant to two or more antimicrobial agents. 8.6% were beta-lactamase producers and accounted for the entire ampicillin-resistant population. CONCLUSION: The prevalence of resistance to penicillin and other commonly used antibiotics among pneumococci is high and the large number of multi-resistant strains among H. influenzae is a cause for concern. The prudent use of antibiotics in treatment of pneumonia and other infections should be advocated to minimise spread of resistance.

Afipia felis induces uptake by macrophages directly into a nonendocytic compartment.

Afipia felis is a Gram-negative bacterium that causes some cases of human Cat Scratch Disease. A. felis can survive and multiply in several mammalian cell types, including macrophages, but the precise intracellular compartmentalization of A. felis-containing phagosomes is unknown. Here, we demonstrate that, in murine macrophages, most A. felis-containing phagosomes exclude lysosomal tracer loaded into macrophage lysosomes before, as well as endocytic tracer loaded after, establishment of an infection. Established Afipia-containing phagosomes possess neither early endosomal marker proteins [early endosome antigen 1 (EEA1), Rab5, transferrin receptor, trytophane aspartate containing coat protein (TACO)] nor late endosomal or lysosomal proteins [cathepsin D, beta-glucuronidase, vacuolar proton-pumping ATPase, rab7, mannose-6-phosphate receptor, vesicle-associated membrane protein 8, lysosome-associated membrane proteins LAMP-1 and LAMP-2]. Those bacteria that will be found in a nonendosomal compartment enter the macrophage via an EEA1-negative compartment, which remains negative for LAMP-1. The smaller subpopulation of afipiae whose phagosomes will be part of the endocytic system enters into an EEA1-positive compartment, which also subsequently acquires LAMP-1. Killing of Afipia or opsonization with immune antibodies leads to a strong increase in the percentage of A. felis-containing phagosomes that interact with the endocytic system. We conclude that most phagosomes containing A. felis are disconnected from the endosome-lysosome continuum, that their unusual compartmentalization is decided at uptake, and that this compartmentalization requires bacterial viability.

Interaction of Listeria monocytogenes with the intestinal epithelium.

Listeria monocytogenes is a food-borne pathogen that must cross the intestinal epithelial barrier to reach its target organs. We have investigated the importance of M cells in translocation using an experimental mouse model and a novel, recently described in vitro coculture system that mimics the follicle-associated epithelium (FAE). Our data demonstrate that L. monocytogenes does not require, nor specifically use, M cells of the FAE to cross the gut. We also show that bacterial translocation is rapid and L. monocytogenes can attach very efficiently to exposed basal lamina of the small intestine indicating an important role for extracellular matrix proteins.

A novel PrfA-regulated chromosomal locus, which is specific for Listeria ivanovii, encodes two small, secreted internalins and contributes to virulence in mice.

Several large, cell wall-associated internalins and one small, secreted internalin (InlC) have been described previously in Listeria monocytogenes. Using degenerate primers derived from sequenced peptides of an L. ivanovii major secreted protein, we identified a new 4.25 kb internalin locus of L. ivanovii, termed i-inlFE. The two proteins encoded by this locus, i-InlE and i-InlF, belong to the group of small, secreted internalins. Southern blot analyses show that the i-inlFE locus does not occur in L. monocytogenes. These data also indicate that six genes encoding small, secreted internalins are present in L. ivanovii, in contrast to L. monocytogenes, in which inlC encodes the only small internalin. The mature i-InlE protein (198 amino acids) is secreted in large amounts into the brain-heart infusion (BHI) culture medium in the stationary growth phase. In minimum essential medium (MEM), which has been used previously to induce PrfA-dependent gene transcription, i-inlE mRNA and i-InlE protein are expressed at high levels. As shown by Northern blot analysis and primer extension, transcription of the tandemly arranged i-inlF and i-inlE genes is dependent on the virulence regulator PrfA, and characteristic palindromic sequences ('PrfA-boxes') were identified in the promoter regions of i-inlF and i-inlE. Non-polar i-inlE and i-inlF deletion mutants and an i-inlFE double deletion mutant were constructed and tested in the mouse infection model. After intravenous infection, all three mutants entirely failed to kill C57BL/6 mice even at high infectious doses of 109 bacteria per mouse, whereas the LD50 for the parental strain was determined as 4 x 107 bacteria per mouse. These data suggest an important role for i-InlE and i-InlF in L. ivanovii virulence.

The gene slyA of Salmonella typhimurium is required for destruction of M cells and intracellular survival but not for invasion or colonization of the murine small intestine.

Recent studies have shown that Salmonella typhimurium invades the M cells of Peyer's patches (PP) of the murine ileum. The slyA gene of S. typhimurium has also recently been reported to affect virulence of this pathogen in mice and survival in macrophages. We therefore compared the effect on PP tissue of four strains of S. typhimurium: a wild-type strain, two slyA insertion mutants, and a recombinant S. typhimurium derivative carrying multiple copies of slyA. Invasion assays performed 2 and 7 days after orogastric infection revealed significantly lower numbers of bacteria of the slyA mutants and of the SlyA-overproducing strain in PP than of the wild type. However, similar numbers of bacteria of all strains were still present in the lumen of the small intestine after these times. Invasion assays of PP tissue after 90-min ileal loop infection yielded comparable numbers of bacteria of all strains in PP. Transmission and scanning electron microscopy of PP tissue after ileal loop infection demonstrated that the two slyA mutants and the SlyA-overproducing strain were able to attach to, induce membrane ruffling of, and invade M cells in a way morphologically and quantitatively similar to that of the wild type. In contrast to the wild type, both slyA mutants and, to a lesser extent, the SlyA-overproducing strain were significantly impaired in their ability to destroy M cells and adjacent enterocytes. Taken together, these data suggest that slyA is involved in intracellular survival and M-cell cytotoxicity but not in the invasion process and that the amount of SlyA needs to be precisely balanced for virulence.