**4.1 For** *Staphylococcus aureus* **MRSA**

Most of the patients can prone to serious bacterial infections caused mainly by the multiresistant microorganisms, *Staphylococcus aureus* coagulase negative spp are one of them. These coagulase negative strains (MRSA) have got approximately 90% of methicillin resistance due to β-lactam resistance (Silva et al., 2011). Story just not stopped here, but still it continues, some of the staphylococcal strains got resistance to the other drugs such as Vancomycin, which was previously widely used against the MRSA infections, and to treat

The Natural Antimicrobial Chromogranins/Secretogranins-Derived

bacterial cells and thus to reach its site of action.

**5. Chromogranin A, a new marker of severity** 

endogenous cascades, cellular dysfunction and death (Baue et al., 1998).

quartile 3 was also significantly different from that of CGA quartile 1 (*P*= 0.033).

Peptides – Production, Lytic Activity and Processing by Bacterial Proteases 197

In conclusion these studies show that CGA-derived AMPs potentiate the effects of antibiotic drugs. One could imagine a mechanism in which the peptide would favor the destabilization of the membrane allowing the antibiotic to rapidly penetrate inside the

In clinical practice, CGA has been used as a marker of pheochromocytomas (O'Connor et al., 1984), carcinoid tumors (O'Connor & Deftos, 1986; Syversen et al., 1993), neuroblastomas (Hsiao et al., 1990), neuroendocrine tumors (Berruti et al., 2005), and neurodegenerative diseases (Rangon et al., 2003). Recent data have shown CGA to be a useful prognostic indicator in patients with chronic heart failure (Omland et al., 2003, suggesting that CGA may have some association with cardiovascular diseases. Furthermore, a pilot study has shown CGA to be a predictor of mortality in patients with acute myocardial infarction (Estensen et al., 2006). Characterization of the severity of organ failures and prediction of patient outcome are of major importance for physicians who care for critically ill patients. Multiple organ failure (MOF) remains the main problem in intensive care because of its impact on morbidity, mortality, and resources (Baue et al., 1998). MOF can develop as a consequence of multiple causes, such as infection, systemic inflammatory response syndrome (SIRS), myocardial infarction, septic shock, leading to the activation of various

In a recent study we have evaluated whether unselected critically ill patients at ICU (Hautepierre Hospital, Strasbourg, France) admission demonstrate increased plasma CGA concentrations and whether CGA can be of any interest in the care of patients at high risk of death. Patients older than 18 years were recruited consecutively over 3 months during 2007. Exclusion criteria included: duration of stay >24 h and conditions known to increase CGA concentrations independently of acute stress [i.e., a history of documented neuroendocrine tumors (O'Connor & Deftos, 1986) or chronic treatment with proton pump inhibitors before admission (Giusti et al., 2004)*.* Patients who required surgical interventions were also excluded. Of the 120 participants included in the study, 70 patients had a primary diagnosis severe infection, and 50 had a SIRS. Serum CGA concentrations were measured with a commercial sandwich RIA kit (a gift of Cisbio Bioassays, Marcoule, France. In the central 95% of the healthy population, serum CGA concentrations range from 19µg/L to 98µg/L. In neuroendocrine system tumors, the CGA serum concentration varies from the typical range up to 1200 µg/L, depending on the biological and structural characteristics of the tumor, as well as on the extent of tumor spread (Degorce et al., 1999). As a control Procalcitonin (PCT) concentrations were measured on the Kryptor system (Brahms Diagnostic) with the timeresolved amplified cryptate emission methodology in accordance with the manufacturer's recommendations. The Simplified Acute Physiological Score II (SAPS II) and the Logistic Organ Dysfunction System (LODS) score were calculated at admission according to published standards (Levy et al., 2003; Le Gall et al., 1993)*.* Our data show that CGA concentration was positively but weakly correlated with age, PCT concentration, creatinine concentration, SAPS II, and LODS score (*P* <0.001 for all variables) and was correlated with CRP concentration (Zhang et al., 2008). Thirty-three deaths occurred during the median follow- up time of 23 days. The death rates for CGA and PCT are shown by quartiles in Figure 7. Statistical analysis revealed a significant difference in death rates between CGA quartile 4 and CGA quartiles 1, 2, and 3 (*P* < 0.001, log-rank test). The death rate for CGA

infections of central nervous system, bone infections and sometimes for the pulmonary infections which require a more concentrations to get treated (Dehority, 2010). *S. aureus* have also developed resistance to the Vancomycin due to the use at low level concentrations and recently, *S. aureus* was isolated that had got the *VanA* gene from the *Enterococcus spp* ( Sievert et al., 2008) which leads to drug resistance.

In our group, we have examined the synergically effects of three CGA-derived peptides (CAT, CTL and CHR) with Minocyclin, Amoxicillin and Linezolide. To demonstrate that antimicrobial peptides are able to reduce the doses of antibiotics used and to potentiate the activity of antibiotics, antimicrobial tests were carried out by combining the antibiotic peptides at doses below the MIC. The comparison was made with the antibiotic or peptide separately at the same doses.

Minocyclin has a MIC of 2 µg/ml alone against the *S. aureus* ATCC 49775, but when it was combined with CTL at a concentration corresponding to 75% of the MIC, the, concentration of Minocycline was lowered to 0.5 µg/ml. Similar data were obtained by the use of the two others peptides (Figure 6). Thus we demonstrate that amidated bCTL acts synergistically with Minocycline against *S. aureus*. In addition CTL acts synergistically with Voriconazole against *Candida albicans* and *Candida tropicalis.*

Fig. 6. Fractional inhibitory concentration (FIC) of the chromogranin derived peptides combined with the antimicrobials (Minocycline against the *Staphylococcus aureus* and Voriconazole against *Candida albicans* and *Candida tropicalis*). FIC in range of ≤0.5 gives a synergistic effect, ≤0.5 - <2 is an additive effect but if more than 2 have an antagonistic effect.

#### **4.2 From** *Shigella*

Some of the strains of *Shigella* got resistance to antibiotics. A 9-year study of shigellosis in Malaysia, show that 58.4% of the studied strains were resistant to tetracyclin and 53.8% to trimetropin-sulfamethoxasol (Banga Singh et al., 2011). In China, another study establish for *Shigella* the resistance to aztrenam (30,8%), ampicillin (92,3%), piperacilline (61,5%), ceftazidime (30,8%), cefotaxime (30,8%), gentamicine (53,8%) (Zhang et al., 2011). Furthermore, *Vibrio cholera* was also described to develop several resistances against antibiotics (Lamrani et al., 2010).

In conclusion these studies show that CGA-derived AMPs potentiate the effects of antibiotic drugs. One could imagine a mechanism in which the peptide would favor the destabilization of the membrane allowing the antibiotic to rapidly penetrate inside the bacterial cells and thus to reach its site of action.
