Staphylococcus aureus Does Not Synthesize Arginine from Proline under Physiological Conditions.

The Gram-positive pathogen Staphylococcus aureus is the only bacterium known to synthesize arginine from proline via the arginine-proline interconversion pathway despite having genes for the well-conserved glutamate pathway. Since the proline-arginine interconversion pathway is repressed by CcpA-mediated carbon catabolite repression (CCR), CCR has been attributed to the arginine auxotrophy of S. aureus. Using ribose as a secondary carbon source, here, we demonstrate that S. aureus arginine auxotrophy is not due to CCR but due to the inadequate concentration of proline degradation product. Proline is degraded by proline dehydrogenase (PutA) into pyrroline-5-carboxylate (P5C). Although the PutA expression was fully induced by ribose, the P5C concentration remained insufficient to support arginine synthesis because P5C was constantly consumed by the P5C reductase ProC. When the P5C concentration was artificially increased by either PutA overexpression or proC deletion, S. aureus could synthesize arginine from proline regardless of carbon source. In contrast, when the P5C concentration was reduced by overexpression of proC, it inhibited the growth of the ccpA deletion mutant without arginine. Intriguingly, the ectopic expression of the glutamate pathway enzymes converted S. aureus into arginine prototroph. In an animal experiment, the arginine-proline interconversion pathway was not required for the survival of S. aureus. Based on these results, we concluded that S. aureus does not synthesize arginine from proline under physiological conditions. We also propose that arginine auxotrophy of S. aureus is not due to the CcpA-mediated CCR but due to the inactivity of the conserved glutamate pathway. IMPORTANCE Staphylococcus aureus is a versatile Gram-positive human pathogen infecting various human organs. The bacterium's versatility is partly due to efficient metabolic regulation via the carbon catabolite repression system (CCR). S. aureus is known to interconvert proline and arginine, and CCR represses the synthesis of both amino acids. However, when CCR is released by a nonpreferred carbon source, S. aureus can synthesize proline but not arginine. In this study, we show that, in S. aureus, the intracellular concentration of pyrroline-5-carboxylate (P5C), the degradation product of proline and the substrate of proline synthesis, is too low to synthesize arginine from proline. These results call into question the notion that S. aureus synthesizes arginine from proline.

Establishing in vitro and in vivo Co-culture Models of Staphylococcus epidermidis and Enterococcus faecalis to Evaluate the Effect of Topical Fluoroquinolone on Ocular Microbes.

Purpose: To establish in vitro and in vivo ocular co-culture models of Staphylococcus epidermidis and Enterococcus faecalis and to study how various concentrations of moxifloxacin affect the survival of these two endophthalmitis-causing bacteria. Methods: Standard strains of S. epidermidis and E. faecalis were used. Color detection agar plates were employed to distinguish their colonies. To establish the in vitro and in vivo co-culture models, S. epidermidis and E. faecalis were co-cultivated at different ratios for various periods. For the in vivo model, various volumes and concentrations of either a mono-culture or co-culture were inoculated into the lower conjunctival sac of rabbits. Finally, the newly developed in vitro and in vivo co-culture models were subjected to the moxifloxacin treatment to access its effect on S. epidermidis and E. faecalis. Results: When S. epidermidis and E. faecalis were cultured separately in tryptic soy broth, their growth peaked and plateaued at approximately 16 and 6 h, respectively. When they were co-cultured, the growth peak of S. epidermidis got delayed, whereas the growth peak of E. faecalis did not change. The number of E. faecalis was significantly higher in the co-culture than that in the mono-culture. Treatment with moxifloxacin in the in vitro co-culture model rapidly decreased the number of S. epidermidis cells at doses ≥ 0.125 µg/ml. In contrast, the number of E. faecalis did not change significantly up to 16 µg/ml moxifloxacin. In in vivo co-culture (at 1:1), the S. epidermidis count decreased in a pattern similar to that seen in in vivo mono-culture and was barely detectable at 24 h after inoculation. In contrast, the of E. faecalis count increased up to 16 h and then decreased. When moxifloxacin was applied (zero, one, or two times) to this model, the S. epidermidis count decreased in proportion to the number of treatments. In contrast, the E. faecalis count increased with moxifloxacin treatment. Conclusions: The in vitro and in vivo co-culture models of S. epidermidis and E. faecalis were established to determine the influence of moxifloxacin eye drops on these bacteria. The results clearly show that the moxifloxacin eye drops can make E. faecalis dominant on the ocular surface.

Fluoroquinolone resistance of Staphylococcus epidermidis isolated from healthy conjunctiva and analysis of their mutations in quinolone-resistance determining region.

BACKGROUND: Staphylococcus epidermidis is the most common pathogen in postoperative endophthalmitis and causes various infectious eye diseases. However, there is very little information on fluoroquinolone antibiotic resistance to S. epidermidis identified in conjunctival microbe and analysis of related genes. Here, the authors investigated the rate of resistance to fluoroquinolones of Staphylococcus epidermidis isolated from normal conjunctival microbes and mutations in the quinolone-resistance determining region (QRDR). METHODS: 377 eye samples from 187 patients who underwent intravitreal injection and cataract surgery were included. Specimens were taken from the bilateral lower conjunctival sacs using a cotton swab and cultured. The cultures were identified using MALDI-TOP MS and gyrA, gyrB, parC, and parE gene mutations of QRDR were confirmed by DNA extraction from resistant strains of S. epidermidis with a micro-dilution method using ciprofloxacin, levofloxacin, and moxifloxacin. RESULTS: The culture positive rate was 61.8% (231) for 374 eye samples. Of the 303 total strains cultured, S. epidermidis was the most common with 33.7% (102). Ten types of gene mutations were observed in the resistant S. epidermidis of 21 strains. One-point mutation was observed mainly in gyrA and parC, and a small number of mutations were observed in parE in the form of a double point mutations. When there were multiple point mutations in both gyrA and parC, the highest minimum inhibitory concentration was observed. CONCLUSIONS: The quinolone resistance rate of S. epidermidis increased in comparison with previous studies, and resistant S. epidermidis showed mostly QRDR mutations, which were mainly found in gyrA and parC, and showed strong resistance when mutated in both genes.

Emerging Enterococcus isolates in postoperative endophthalmitis by selection pressure of fluoroquinolones: an 11-year multicenter and experimental study.

Postoperative endophthalmitis (PE) is the devastating complication that frequently results in vision loss. Recently, enterococcus have emerged as a major cause of PE in several countries and resulted in poor visual outcome. However, the reason remains elusive. We investigate whether selection pressure of fluoroquinolone exerts effects on microorganism profiles isolated from PE. Medical records of patients who were diagnosed with PE at eight resident training institutions between January 2004 and December 2015 were reviewed. The most common isolate was Enterococcus faecalis (28.0%), followed by Staphylococcus epidermidis (18.6%) and other coagulase negative Staphylococci (7.6%). However, the rates of E. faecalis isolated from conjunctival microbes were 6.2% (16/257) and their resistance to fluoroquinolones was higher than those of S. epidermidis. In vitro and in vivo co-culture models of E. faecalis and S. epidermidis were established for survival assays after administration of fourth-generation fluoroquinolone. In in vitro co-culture model, the survival assay of E. faecalis and S. epidermidis against the treatment of moxifloxacin showed that E. faecalis survived significantly better than S. epidermidis in the presence of moxifloxacin 1 µg/mL and more. In in vivo co-culture model, E. faecalis survived significantly better than S. epidermidis after topical treatment of moxifloxacin (5 mg/mL). E. faecalis has been the most common causative strain of PE in Korea. We suggest that the increase of E. faecalis in PE could be associated with the selection pressure of fourth-generation fluoroquinolone. Summary: Enterococcus spp. have emerged as a leading causative strain of postoperative endophthalmitis in 11-year clinical data. We suggest that the increase of Enterococcus spp. is associated with the selection pressure of fourth-generation fluoroquinolone.

Factors affecting precipitation of vancomycin and ceftazidime on intravitreal injection.

PURPOSE: The aim of this study was to investigate the factors that affect the precipitation of vancomycin and ceftazidime which are currently used in intravitreal injections for infectious endophthalmitis. METHODS: The minimum concentration required for precipitation of the 2 antibiotics was estimated by 2-fold dilution. In addition, the amount of the precipitate that formed was measured at 4, 3, 2, and 1 mL volumes, temperatures (37°C or 4°C), and in different medias (balanced salt solution plus and normal saline). RESULTS: When 2-fold serial dilutions of vancomycin and ceftazidime were mixed, precipitates started forming at a concentration of 12.5 mg/mL for ceftazidime and 10 mg/mL for vancomycin. The precipitation of vancomycin and ceftazidime increased at lower temperatures (4°C) and lower media volumes (1 mL). However, using a micropipette with a complete mixed media and an antibiotic (vancomycin or ceftazidime) prevented the formation of a precipitate when the other antibiotic was added. CONCLUSION: The formation of a precipitate associated with intravitreal injections was correlated with the diffusion rate of the antibiotics in the media. Precipitation increased at lower temperatures, lower media volumes, and at higher antibiotic concentrations.

Effects of ß-lapachone on the production of Th1 and Th2 cytokines in C57BL/6 mice.

In this study, we investigated the effects of ß-lapachone (ß-lap) on the production of cytokines in C57BL/6 mice. The culture supernatants of splenocytes exposed to ß-lap plus lipopolysaccharide or concanavalin A (Con A) were harvested to determine Th1 (tumor necrosis factor-α, interferon-γ, interleukin [IL]-1ß, IL-2, IL-17A, and IL-12) and Th2 (IL-4, IL-5, IL-6, and IL-10) cytokines. IL-2, a Th1 cytokine, was expressed at higher levels in splenocytes treated with ß-lap and with lipopolysaccharide plus ß-lap. Expression of Th2 cytokines, including IL-4, IL-5, IL-6, and IL-10, was lower in the culture supernatants of mouse splenocytes exposed to Con A plus ß-lap than in supernatants from cells that were exposed to Con A alone. These findings demonstrate that ß-lap downregulates the immediate hypersensitivity reaction induced by Con A. Therefore, Th2 responses involve the downregulation of IL-4, IL-5, IL-6, and IL-10, preventing the occurrence of allergies and various manifestations of allergic inflammation. These results suggest that ß-lap may have potential preventive or adjunct anti-inflammatory therapy applications because of its function in modulating the production of cytokines. However, further in vivo investigations of this process are necessary to elucidate the mechanisms involved.

A change in PBP1 is involved in amoxicillin resistance of clinical isolates of Helicobacter pylori.

Reports on the isolation of amoxicillin-resistant Helicobacter pylori are increasing worldwide, which may cause serious problems in eradication therapy. To elucidate the mechanism of amoxicillin resistance of H. pylori, penicillin-binding proteins (PBPs) of amoxicillin-resistant strains isolated in Korea were analysed. Three PBPs (66, 63 and 60 kDa) were identified in both amoxicillin-resistant and -susceptible strains using biotinylated ampicillin, and the PBP profiles were very similar irrespective of the difference in amoxicillin susceptibility. We obtained clones with moderate resistance from an amoxicillin-susceptible strain, HPK5, by transformation with genomic DNA from an amoxicillin-resistant strain, HPA116. In a resistance-induced clone, HPO1, the affinity of PBP1 for amoxicillin was reduced. The pbp1 genes from HPA116, HPO1 and HPK5 were cloned and sequenced. The nucleotide sequences of pbp1 from HPA116 and HPO1 were almost identical, whereas that of HPK5 was quite different. Both the ORFs of HPA116 and HPO1 pbp1 have four substitutions and one insertion of amino acid residues compared with those of HPK5 and other sensitive strains. All the mutations, except one, are in the C-terminal half of the 659-amino-acid sequence containing the penicillin-binding modules. DNA fragments containing either full-length or a C-terminal half of pbp1 could transform HPK5 to have resistance, indicating that changes in the penicillin-binding core of PBP1 are involved in the amoxicillin resistance of H. pylori isolated in Korea.