Background Methicillin resistant  Staphylococcus aureus  (MRSA) is the leading pathogenic cause of nosocomial infections, especially in bacteraemia and sepsis. The essential therapy for MRSA infection is glycopeptides. Therapeutic failure can be seen with this therapy and the mortality is still high. The aim of this study was to evaluate the additional effect of G-CSF on the traditional antibiotic treatment in an experimental MRSA sepsis.
Background
Methicillin resistant  Staphylococcus aureus  (MRSA) is the leading pathogenic cause of nosocomial infections, especially in bacteraemia and sepsis. The essential therapy for MRSA infection is glycopeptides. Therapeutic failure can be seen with this therapy and the mortality is still high. The aim of this study was to evaluate the additional effect of G-CSF on the traditional antibiotic treatment in an experimental MRSA sepsis.Staphylococcus aureusMethods Experimental sepsis was performed in mice by intraperitoneal injection of MRSA isolate. Inoculum dose was estimated as 6 × 10 9 /ml. Mice were randomised for the study into four group; control group (not receive any therapy), G-CSF group (1000 ng/daily, subcutaneously for 3 d), antibiotic group (vancomycin 25 or 50 mg/kg intraperitoneally every 12 hours for 7 d), and vancomycin+G-CSF group (at the same concentrations and duration). Autopsy was done within one hour after mice died. If mice was still alive at the end of seventh day, they were sacrificed, and autopsy was done. In all groups, the effect of G-CSF therapy on the survival, the number of the MRSA colonies in the lung, liver, heart, spleen, and peritoneal cultures, the histopathology of the lung, liver, heart and spleen was investigated.
Methods
Experimental sepsis was performed in mice by intraperitoneal injection of MRSA isolate. Inoculum dose was estimated as 6 × 10 9 /ml. Mice were randomised for the study into four group; control group (not receive any therapy), G-CSF group (1000 ng/daily, subcutaneously for 3 d), antibiotic group (vancomycin 25 or 50 mg/kg intraperitoneally every 12 hours for 7 d), and vancomycin+G-CSF group (at the same concentrations and duration). Autopsy was done within one hour after mice died. If mice was still alive at the end of seventh day, they were sacrificed, and autopsy was done. In all groups, the effect of G-CSF therapy on the survival, the number of the MRSA colonies in the lung, liver, heart, spleen, and peritoneal cultures, the histopathology of the lung, liver, heart and spleen was investigated.Results One hundred and six mice were used. There were no significant differences in survival rates and bacterial eradication in G-CSF group compared with control group, and also in antibiotic +G-CSF group compared with antibiotic alone group. These parameters were all significantly different in antibiotic alone group compared with control group. Histopathologically, inflammation of the lung and liver were significantly reduced in vancomycin (25 mg/kg)+G-CSF and vancomycin (50 mg/kg)+G-CSF subgroups, respectively (p < 0.01). The histopathological inflammation of the other organs was not significantly different in antibiotic+G-CSF group compared with antibiotic group and, also G-CSF group compared with control group.
Results
One hundred and six mice were used. There were no significant differences in survival rates and bacterial eradication in G-CSF group compared with control group, and also in antibiotic +G-CSF group compared with antibiotic alone group. These parameters were all significantly different in antibiotic alone group compared with control group. Histopathologically, inflammation of the lung and liver were significantly reduced in vancomycin (25 mg/kg)+G-CSF and vancomycin (50 mg/kg)+G-CSF subgroups, respectively (p < 0.01). The histopathological inflammation of the other organs was not significantly different in antibiotic+G-CSF group compared with antibiotic group and, also G-CSF group compared with control group.Conclusion G-CSF treatment had no additional effect on survival and bacterial eradication in MRSA sepsis in nonneutropenic mice; and only a little effect on histopathology. G-CSF treatment is very expensive, likewise glycopeptides. The more interest in infection control measures, and prevent the spread of MRSA infections is more rational.
Conclusion
G-CSF treatment had no additional effect on survival and bacterial eradication in MRSA sepsis in nonneutropenic mice; and only a little effect on histopathology. G-CSF treatment is very expensive, likewise glycopeptides. The more interest in infection control measures, and prevent the spread of MRSA infections is more rational.

Background Staphylococcus aureus  is an extremely virulent pathogen, and causes serious and deep-seated infections (e.g. endocarditis, osteomyelitis) [ 1 ]. In recent years,  S. aureus  is the leading pathogenic cause of both community-acquired and nosocomial infections, especially in bacteremia and sepsis [ 2 - 4 ]. Despite advances in antistaphylococcal drugs,  S. aureus  sepsis is one of the most important causes of death [ 5 ]. Furthermore, methicillin resistant strains are increasing in community and hospitals during the past decades, and many investigators proposed methicillin resistance as an independent predictor of adverse outcome [ 6 , 7 ]. The essential therapy for methicillin resistant  S. aureus  (MRSA) infections is glycopeptides (vancomycin or teicoplanin). However glycopeptides are intrinsically less effective against staphylococci than are antistaphyloccal β-lactams [ 1 ]. This may explain therapeutic failure and high mortality of MRSA infections. Advances in the pathophysiology of sepsis and septic shock suggest new therapeutic agents and approaches. Granulocyte-colony stimulating factor (G-CSF), a potent stimulator of neutrophil counts and functions, is one of these new strategies in sepsis. Despite a number of studies in sepsis, especially in neonatal sepsis, clinical efficacy of G-CSF is still controversial [ 8 ]. The aim of this experimental study was to evaluate the role of G-CSF in the treatment of MRSA sepsis.
Background
Staphylococcus aureus  is an extremely virulent pathogen, and causes serious and deep-seated infections (e.g. endocarditis, osteomyelitis) [ 1 ]. In recent years,  S. aureus  is the leading pathogenic cause of both community-acquired and nosocomial infections, especially in bacteremia and sepsis [ 2 - 4 ]. Despite advances in antistaphylococcal drugs,  S. aureus  sepsis is one of the most important causes of death [ 5 ]. Furthermore, methicillin resistant strains are increasing in community and hospitals during the past decades, and many investigators proposed methicillin resistance as an independent predictor of adverse outcome [ 6 , 7 ]. The essential therapy for methicillin resistant  S. aureus  (MRSA) infections is glycopeptides (vancomycin or teicoplanin). However glycopeptides are intrinsically less effective against staphylococci than are antistaphyloccal β-lactams [ 1 ]. This may explain therapeutic failure and high mortality of MRSA infections.Staphylococcus aureusS. aureusS. aureusS. aureusAdvances in the pathophysiology of sepsis and septic shock suggest new therapeutic agents and approaches. Granulocyte-colony stimulating factor (G-CSF), a potent stimulator of neutrophil counts and functions, is one of these new strategies in sepsis. Despite a number of studies in sepsis, especially in neonatal sepsis, clinical efficacy of G-CSF is still controversial [ 8 ].The aim of this experimental study was to evaluate the role of G-CSF in the treatment of MRSA sepsis.Methods Animal care and use Male BALB-C mice (8–10 weeks old, weighing 20–25 g) were obtained from Erciyes University Hakan Cetinsaya Experimental and Clinical Research Center. The Animal Care Committee of Erciyes University approved the experimental protocol used in this study. The animals were kept in a cage and allowed to feed and drink water. Bacterial strain The clinical MRSA isolate, obtained from a patient's blood and pleural effusion admitted in General Surgical Intensive Care Unit of Erciyes University with nosocomial sepsis, was used. The strain was subcultured on blood agar at 37°C over night. On the day of experiment, bacterial suspension was prepared by sodium chloride 0.9% solution and the concentration was adjusted by spectrophotometer. The bacterial account needed for experimental sepsis was determined by a fore study. The fore study was begun with the inoculum dose of 1 × 10 7 /ml bacterial suspension and gradually increased until experimental sepsis developed. Experimental sepsis was defined as the growth of MRSA in two or more organs. The inoculum dose of this study was estimated as 6 × 10 9 /mL. Treatment First dose of antibiotic was given at the sixth hour of bacterial inoculation. Vancomycin (DBL, 500 mg flacon), diluted in 5% dextrose, was given at 25 mg/kg or 50 mg/kg concentrations intraperitoneally every 12 hours for 7 days. rhG-CSF (Roche, Neupogen flk 30 mu/ 1 mL), diluted in 5% dextrose, was received 1000 ng/daily subcutaneously for 3 days [ 9 ]. Study design Animals were randomised to four groups; control group, G-CSF group, vancomycin group, and vancomycin +G-CSF group. The last two groups were divided into two subgroups for different dosage of vancomycin (25 mg/kg or 50 mg/kg). Each group had at least 15 mice and were kept in different cage. In control group, only bacteria suspension was given and no treatment was received. Bacterial suspension was given intraperitoneally to the mice, and when mice died, autopsy was done within one hour in aseptic conditions. If the mice was still alive at the end of seventh day, mice were sacrificed by servical dislocation and autopsy was done. The samples were taken from lung, liver, heart, spleen, and peritoneum for microbiological investigation, and from lung, liver, heart and spleen for histopathological examination. In each group, survival days were noted. Samples were taken from the organs with swab by one rotation on its axis, and were cultured on blood agar over night. The colonies on the agar were counted. The colonies more than 300 cfu in a plate noted as >300 cfu. The same pathologist examined tissue samples stained with hematoxylin and eosin. The degree of inflammation was graded by on a scale of 0 to ++++ (0, no inflammation; focal interstitial inflammation +; more diffuse interstitial inflammation ++; intense interstitial inflammation or microabscesses +++; more extensive abscess formation with tissue necrosis ++++) [ 10 ]. The pathologist was unaware about the groups. Survival days, semiquantitative bacterial count and histopathologic findings in the tissues of the treatment groups were compared with the control group. Statistics Survival was assessed using a log-rank test. Fisher's extract test and chi-square test were used to compare the groups. Mann Whitney U test was used to compare the histopathology. A p value less than 0.05 was considered significant.
Methods
Animal care and use Male BALB-C mice (8–10 weeks old, weighing 20–25 g) were obtained from Erciyes University Hakan Cetinsaya Experimental and Clinical Research Center. The Animal Care Committee of Erciyes University approved the experimental protocol used in this study. The animals were kept in a cage and allowed to feed and drink water.
Animal care and use
Male BALB-C mice (8–10 weeks old, weighing 20–25 g) were obtained from Erciyes University Hakan Cetinsaya Experimental and Clinical Research Center. The Animal Care Committee of Erciyes University approved the experimental protocol used in this study. The animals were kept in a cage and allowed to feed and drink water.Bacterial strain The clinical MRSA isolate, obtained from a patient's blood and pleural effusion admitted in General Surgical Intensive Care Unit of Erciyes University with nosocomial sepsis, was used. The strain was subcultured on blood agar at 37°C over night. On the day of experiment, bacterial suspension was prepared by sodium chloride 0.9% solution and the concentration was adjusted by spectrophotometer. The bacterial account needed for experimental sepsis was determined by a fore study. The fore study was begun with the inoculum dose of 1 × 10 7 /ml bacterial suspension and gradually increased until experimental sepsis developed. Experimental sepsis was defined as the growth of MRSA in two or more organs. The inoculum dose of this study was estimated as 6 × 10 9 /mL.
Bacterial strain
The clinical MRSA isolate, obtained from a patient's blood and pleural effusion admitted in General Surgical Intensive Care Unit of Erciyes University with nosocomial sepsis, was used. The strain was subcultured on blood agar at 37°C over night. On the day of experiment, bacterial suspension was prepared by sodium chloride 0.9% solution and the concentration was adjusted by spectrophotometer. The bacterial account needed for experimental sepsis was determined by a fore study. The fore study was begun with the inoculum dose of 1 × 10 7 /ml bacterial suspension and gradually increased until experimental sepsis developed. Experimental sepsis was defined as the growth of MRSA in two or more organs. The inoculum dose of this study was estimated as 6 × 10 9 /mL.Treatment First dose of antibiotic was given at the sixth hour of bacterial inoculation. Vancomycin (DBL, 500 mg flacon), diluted in 5% dextrose, was given at 25 mg/kg or 50 mg/kg concentrations intraperitoneally every 12 hours for 7 days. rhG-CSF (Roche, Neupogen flk 30 mu/ 1 mL), diluted in 5% dextrose, was received 1000 ng/daily subcutaneously for 3 days [ 9 ].
Treatment
First dose of antibiotic was given at the sixth hour of bacterial inoculation. Vancomycin (DBL, 500 mg flacon), diluted in 5% dextrose, was given at 25 mg/kg or 50 mg/kg concentrations intraperitoneally every 12 hours for 7 days. rhG-CSF (Roche, Neupogen flk 30 mu/ 1 mL), diluted in 5% dextrose, was received 1000 ng/daily subcutaneously for 3 days [ 9 ].Study design Animals were randomised to four groups; control group, G-CSF group, vancomycin group, and vancomycin +G-CSF group. The last two groups were divided into two subgroups for different dosage of vancomycin (25 mg/kg or 50 mg/kg). Each group had at least 15 mice and were kept in different cage. In control group, only bacteria suspension was given and no treatment was received. Bacterial suspension was given intraperitoneally to the mice, and when mice died, autopsy was done within one hour in aseptic conditions. If the mice was still alive at the end of seventh day, mice were sacrificed by servical dislocation and autopsy was done. The samples were taken from lung, liver, heart, spleen, and peritoneum for microbiological investigation, and from lung, liver, heart and spleen for histopathological examination. In each group, survival days were noted. Samples were taken from the organs with swab by one rotation on its axis, and were cultured on blood agar over night. The colonies on the agar were counted. The colonies more than 300 cfu in a plate noted as >300 cfu. The same pathologist examined tissue samples stained with hematoxylin and eosin. The degree of inflammation was graded by on a scale of 0 to ++++ (0, no inflammation; focal interstitial inflammation +; more diffuse interstitial inflammation ++; intense interstitial inflammation or microabscesses +++; more extensive abscess formation with tissue necrosis ++++) [ 10 ]. The pathologist was unaware about the groups. Survival days, semiquantitative bacterial count and histopathologic findings in the tissues of the treatment groups were compared with the control group.
Study design
Animals were randomised to four groups; control group, G-CSF group, vancomycin group, and vancomycin +G-CSF group. The last two groups were divided into two subgroups for different dosage of vancomycin (25 mg/kg or 50 mg/kg). Each group had at least 15 mice and were kept in different cage. In control group, only bacteria suspension was given and no treatment was received.Bacterial suspension was given intraperitoneally to the mice, and when mice died, autopsy was done within one hour in aseptic conditions. If the mice was still alive at the end of seventh day, mice were sacrificed by servical dislocation and autopsy was done. The samples were taken from lung, liver, heart, spleen, and peritoneum for microbiological investigation, and from lung, liver, heart and spleen for histopathological examination. In each group, survival days were noted.Samples were taken from the organs with swab by one rotation on its axis, and were cultured on blood agar over night. The colonies on the agar were counted. The colonies more than 300 cfu in a plate noted as >300 cfu.The same pathologist examined tissue samples stained with hematoxylin and eosin. The degree of inflammation was graded by on a scale of 0 to ++++ (0, no inflammation; focal interstitial inflammation +; more diffuse interstitial inflammation ++; intense interstitial inflammation or microabscesses +++; more extensive abscess formation with tissue necrosis ++++) [ 10 ]. The pathologist was unaware about the groups.Survival days, semiquantitative bacterial count and histopathologic findings in the tissues of the treatment groups were compared with the control group.Statistics Survival was assessed using a log-rank test. Fisher's extract test and chi-square test were used to compare the groups. Mann Whitney U test was used to compare the histopathology. A p value less than 0.05 was considered significant.
Statistics
Survival was assessed using a log-rank test. Fisher's extract test and chi-square test were used to compare the groups. Mann Whitney U test was used to compare the histopathology. A p value less than 0.05 was considered significant.Results This study included 110 mice. Three mice that developed intraabdominal hemorrhage, and one mouse that developed  Esherichia coli  sepsis were excluded. 106 mice were evaluated. Six mice in control group, six mice in G-CSF group, 15 mice in vancomycin 25 mg, 15 mice in vancomycin 25 mg+G-CSF group, 18 mice in vancomycin 50 mg group and 14 mice in vancomycin 50 mg+G-CSF group were sacrificed at the end of seventh day. The death day and the survival rate of the mice in the groups are shown by the survival curve in figure  1 , 2 , 3 . The comparison of the groups between G-CSF and control, vancomycin (25 mg/kg) and vancomycin (25 mg/kg)+G-CSF, vancomycin (50 mg/kg) and vancomycin (50 mg/kg)+G-CSF showed no differences (p >0.05). Culture and histopathology results are shown in Table  1 . Antibiotic administration decreased bacterial count, and decreased inflammation rate in the organs. Culture and histopathological results in control and G-CSF group were not statistically different (p > 0.05). However, all these parameters were significantly different in antibiotic groups compared with control group (p < 0.01). Cultures of the organs in antibiotic groups were not statistically different from antibiotic+G-CSF group (p > 0.05). Only the inflammation degree in the lung and the liver were significantly reduced in vancomycin (25 mg/kg)+G-CSF group and vancomycin (50 mg/kg)+G-CSF group, respectively (p < 0.01). The inflammatory changes were not significantly reduced in the other organs in two groups (p > 0.05).
Results
This study included 110 mice. Three mice that developed intraabdominal hemorrhage, and one mouse that developed  Esherichia coli  sepsis were excluded. 106 mice were evaluated. Six mice in control group, six mice in G-CSF group, 15 mice in vancomycin 25 mg, 15 mice in vancomycin 25 mg+G-CSF group, 18 mice in vancomycin 50 mg group and 14 mice in vancomycin 50 mg+G-CSF group were sacrificed at the end of seventh day. The death day and the survival rate of the mice in the groups are shown by the survival curve in figure  1 , 2 , 3 . The comparison of the groups between G-CSF and control, vancomycin (25 mg/kg) and vancomycin (25 mg/kg)+G-CSF, vancomycin (50 mg/kg) and vancomycin (50 mg/kg)+G-CSF showed no differences (p >0.05).Esherichia coliCulture and histopathology results are shown in Table  1 . Antibiotic administration decreased bacterial count, and decreased inflammation rate in the organs. Culture and histopathological results in control and G-CSF group were not statistically different (p > 0.05). However, all these parameters were significantly different in antibiotic groups compared with control group (p < 0.01). Cultures of the organs in antibiotic groups were not statistically different from antibiotic+G-CSF group (p > 0.05). Only the inflammation degree in the lung and the liver were significantly reduced in vancomycin (25 mg/kg)+G-CSF group and vancomycin (50 mg/kg)+G-CSF group, respectively (p < 0.01). The inflammatory changes were not significantly reduced in the other organs in two groups (p > 0.05).Discussion In recent years, MRSA has become widespread around the world, and become highly endemic in some hospitals. In the United States, the proportion of MRSA isolates increased from 2.4% in 1975 to up to 55% in recent years [ 11 , 12 ]. Similarly, in Europe the resistance rates increased from 12.8% to 26.3% [ 12 , 13 ]. Also, it is extremely high (>60%) in some regions of the world [ 12 ]. The recent studies, conducted in our hospital, showed 66% methicillin resistance in nosocomial  S. aureus  strains, isolated from the bloodstream infections [ 2 , 14 ]. Unfortunately, community-acquired MRSA also increases during the past decades [ 15 ]. G-CSF is a cytokine that stimulates myeloid progenitor cell proliferation and increases the bone marrow storage pool and the number of circulating mature neutrophils, which are important component of the host defense [ 16 ]. Also, it enhances neutrophil activities (chemotaxis, phagocytosis, antibody-mediated cytotoxicity, etc.) [ 17 ]. An inappropriate endogenous G-CSF response may be associated with an adverse outcome to sepsis. Low serum G-CSF concentrations (0 to 125 pg/mL) on admission are supposed to be associated with fatal outcome in patients with bacterial infections [ 18 ]. Investigators proposed to use G-CSF in infections in which neutrophil number and function are important to resolution and survival, also in patients which may have reduced neutrophil numbers or function because of underlying disease or physiologic state. The low toxicity and the beneficial effect on survival in animal studies have led to several clinical trials of rhG-CSF as an adjuvant therapy in treatment of infection in nonneutropenic patients [ 19 , 20 ]. Its beneficial effect was shown in clinical studies in diabetic foot infections, wound infections, extensive burns and fungal infections [ 21 - 23 ]. Also neutrophils in sepsis demonstrate a number of functional abnormalities (e.g. reduced bacterial killing, superoxide production, and migration) [ 24 ] and it can be hypothesized that these abnormalities can be corrected with G-CSF. The previous experimental therapeutic studies were mostly carried out in gram-negative sepsis, and in this experimental study, we investigated the effect of G-CSF in MRSA sepsis. The survival rates were not significantly different in G-CSF group compared with the control group, and also in antibiotic+G-CSF group compared with antibiotic alone group. Likewise, most of the other experimental studies in gram negative sepsis, showed that prophylactic rhG-CSF administration reduced endotoxemia and serum TNF-α levels and also improved cardiac function and survival, whereas therapeutic rhG-CSF (i.e. administered after the onset of infection) did not improve outcome and at very high dosages appeared harmful [ 25 , 26 ]. A recent multicenter, double-blind, randomised and placebo controlled study in patients hospitalised with pneumonia and severe sepsis, demonstrated that G-CSF had no beneficial effect in reducing mortality rates or complications from severe sepsis [ 27 ]. In this study, we did not measure the WBC count, so not have subgroups with neutropenia. In an experimental gram-negative sepsis in rabbits, Smith and colleguaes [ 28 ] showed the beneficial effect of therapeutic rhG-CSF only in early sepsis due to gram negative bacteria when complicated by leukopenia, no significant difference in nonneutropenic group. Bacteria are rapidly cleared from blood and tissues following intravenous antibiotic therapy [ 10 ]. In our study, antibiotic received group had significantly low bacterial count in the organ cultures compared with the other groups, however G-CSF had no beneficial effect on the bacterial clearance. The histopathologic findings of invasive  S. aureus  infections are leukocytic infiltration, focal pneumonitis, edema, microabscesses, etc. In an experimental study, significant pathologic changes during and after the elimination of bacteria from the blood and tissues were noted. Also, expression of cytokines (TNF, IL-1, IL-6) was observed in all of the infected tissues, and correlated tissue damage after clearance of bacteria from the blood and tissues[ 10 ]. However, in our study, G-CSF significantly reduced only the inflammation in the lung and liver in vancomycin +G-CSF subgroups (p < 0.01). This effect of G-CSF could not be explained. There was no significant effect of G-CSF on the other organs inflammation. In conclusion, G-CSF treatment had no additional effect on survival and bacterial eradication in MRSA sepsis in nonneutropenic mice; and only a little effect on histopathology. Furthermore, G-CSF treatment is very expensive, likewise glycopeptides. Because of high mortality and morbidity rates and excess costs, more interest in infection control measures, and prevent the spread of MRSA infections is more rational.
Discussion
In recent years, MRSA has become widespread around the world, and become highly endemic in some hospitals. In the United States, the proportion of MRSA isolates increased from 2.4% in 1975 to up to 55% in recent years [ 11 , 12 ]. Similarly, in Europe the resistance rates increased from 12.8% to 26.3% [ 12 , 13 ]. Also, it is extremely high (>60%) in some regions of the world [ 12 ]. The recent studies, conducted in our hospital, showed 66% methicillin resistance in nosocomial  S. aureus  strains, isolated from the bloodstream infections [ 2 , 14 ]. Unfortunately, community-acquired MRSA also increases during the past decades [ 15 ].S. aureusG-CSF is a cytokine that stimulates myeloid progenitor cell proliferation and increases the bone marrow storage pool and the number of circulating mature neutrophils, which are important component of the host defense [ 16 ]. Also, it enhances neutrophil activities (chemotaxis, phagocytosis, antibody-mediated cytotoxicity, etc.) [ 17 ]. An inappropriate endogenous G-CSF response may be associated with an adverse outcome to sepsis. Low serum G-CSF concentrations (0 to 125 pg/mL) on admission are supposed to be associated with fatal outcome in patients with bacterial infections [ 18 ]. Investigators proposed to use G-CSF in infections in which neutrophil number and function are important to resolution and survival, also in patients which may have reduced neutrophil numbers or function because of underlying disease or physiologic state. The low toxicity and the beneficial effect on survival in animal studies have led to several clinical trials of rhG-CSF as an adjuvant therapy in treatment of infection in nonneutropenic patients [ 19 , 20 ]. Its beneficial effect was shown in clinical studies in diabetic foot infections, wound infections, extensive burns and fungal infections [ 21 - 23 ]. Also neutrophils in sepsis demonstrate a number of functional abnormalities (e.g. reduced bacterial killing, superoxide production, and migration) [ 24 ] and it can be hypothesized that these abnormalities can be corrected with G-CSF.The previous experimental therapeutic studies were mostly carried out in gram-negative sepsis, and in this experimental study, we investigated the effect of G-CSF in MRSA sepsis. The survival rates were not significantly different in G-CSF group compared with the control group, and also in antibiotic+G-CSF group compared with antibiotic alone group. Likewise, most of the other experimental studies in gram negative sepsis, showed that prophylactic rhG-CSF administration reduced endotoxemia and serum TNF-α levels and also improved cardiac function and survival, whereas therapeutic rhG-CSF (i.e. administered after the onset of infection) did not improve outcome and at very high dosages appeared harmful [ 25 , 26 ]. A recent multicenter, double-blind, randomised and placebo controlled study in patients hospitalised with pneumonia and severe sepsis, demonstrated that G-CSF had no beneficial effect in reducing mortality rates or complications from severe sepsis [ 27 ].In this study, we did not measure the WBC count, so not have subgroups with neutropenia. In an experimental gram-negative sepsis in rabbits, Smith and colleguaes [ 28 ] showed the beneficial effect of therapeutic rhG-CSF only in early sepsis due to gram negative bacteria when complicated by leukopenia, no significant difference in nonneutropenic group.Bacteria are rapidly cleared from blood and tissues following intravenous antibiotic therapy [ 10 ]. In our study, antibiotic received group had significantly low bacterial count in the organ cultures compared with the other groups, however G-CSF had no beneficial effect on the bacterial clearance.The histopathologic findings of invasive  S. aureus  infections are leukocytic infiltration, focal pneumonitis, edema, microabscesses, etc. In an experimental study, significant pathologic changes during and after the elimination of bacteria from the blood and tissues were noted. Also, expression of cytokines (TNF, IL-1, IL-6) was observed in all of the infected tissues, and correlated tissue damage after clearance of bacteria from the blood and tissues[ 10 ]. However, in our study, G-CSF significantly reduced only the inflammation in the lung and liver in vancomycin +G-CSF subgroups (p < 0.01). This effect of G-CSF could not be explained. There was no significant effect of G-CSF on the other organs inflammation.S. aureusIn conclusion, G-CSF treatment had no additional effect on survival and bacterial eradication in MRSA sepsis in nonneutropenic mice; and only a little effect on histopathology. Furthermore, G-CSF treatment is very expensive, likewise glycopeptides. Because of high mortality and morbidity rates and excess costs, more interest in infection control measures, and prevent the spread of MRSA infections is more rational.Competing interests This study was supported by Erciyes University Research Fund. There was no non-financial competing interest.
Competing interests
This study was supported by Erciyes University Research Fund. There was no non-financial competing interest.Authors' contributions EA and SG were the primary researchers. OC and BK are the pathologist and examined tissue samples. MD was the director of the study. All authors read and approved the final manuscript.
Authors' contributions
EA and SG were the primary researchers. OC and BK are the pathologist and examined tissue samples. MD was the director of the study. All authors read and approved the final manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here:


Background Enterococci tend to be one of the leading causes of nosocomial infections, with  E. faecalis  and  E. faecium  accounting up to 90% of the clinical isolates. Nevertheless, the incidence of other species of enterococci from clinical sources shows an alarming increase with the properties of intrinsic resistance to several antibiotics including beta-lactams and glycopeptides. Thus proper identification of enterococci to species level is quintessential for management and prevention of these bacteria in any healthcare facility. Hence this work was undertaken to study the prevalence of unusual species of enterococci causing human infections, in a tertiary care hospital in South India.
Background
Enterococci tend to be one of the leading causes of nosocomial infections, with  E. faecalis  and  E. faecium  accounting up to 90% of the clinical isolates. Nevertheless, the incidence of other species of enterococci from clinical sources shows an alarming increase with the properties of intrinsic resistance to several antibiotics including beta-lactams and glycopeptides. Thus proper identification of enterococci to species level is quintessential for management and prevention of these bacteria in any healthcare facility. Hence this work was undertaken to study the prevalence of unusual species of enterococci causing human infections, in a tertiary care hospital in South India.E. faecalisE. faeciumMethods The study was conducted in a tertiary care hospital in South India from July 2001 to June 2003. Isolates of enterococci were collected from various clinical specimens and speciated using extensive phenotypic and physiological tests. Antimicrobial susceptibility testing were performed and interpreted as per NCCLS guidelines. Whole cell protein (WCP) fingerprinting of enterococci were done for species validation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed computationally.
Methods
The study was conducted in a tertiary care hospital in South India from July 2001 to June 2003. Isolates of enterococci were collected from various clinical specimens and speciated using extensive phenotypic and physiological tests. Antimicrobial susceptibility testing were performed and interpreted as per NCCLS guidelines. Whole cell protein (WCP) fingerprinting of enterococci were done for species validation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed computationally.Results Our study showed the prevalence of unusual (non-faecalis and non-faecium enterococci) and atypical (biochemical variant) species of enterococci as 19% (46 isolates) and 5% (12 isolates) respectively. The 7 unusual species (46 isolates) isolated and confirmed by phenotypic characterization includes: 15  E. gallinarum  (6.2%), 10  E. avium  (4.1%), 6  E. raffinosus  (2.5%), 6  E. hirae  (2.5%), 4  E. mundtii  (1.7%), 3  E. casseliflavus -including the two atypical isolates (1.2%) and 2  E. durans  (0.8%). The 12 atypical enterococcal species (5%) that showed aberrant sugar reactions in conventional phenotyping were confirmed as  E. faecalis, E. faecium  and  E. casseliflavus  respectively by WCP fingerprinting. The antimicrobial susceptibility testing depicted the emergence of high-level aminoglycoside and beta-lactam resistance among different species apart from intrinsic vancomycin resistance by some species, while all the species tested were susceptible for linezolid and teicoplanin.
Results
Our study showed the prevalence of unusual (non-faecalis and non-faecium enterococci) and atypical (biochemical variant) species of enterococci as 19% (46 isolates) and 5% (12 isolates) respectively. The 7 unusual species (46 isolates) isolated and confirmed by phenotypic characterization includes: 15  E. gallinarum  (6.2%), 10  E. avium  (4.1%), 6  E. raffinosus  (2.5%), 6  E. hirae  (2.5%), 4  E. mundtii  (1.7%), 3  E. casseliflavus -including the two atypical isolates (1.2%) and 2  E. durans  (0.8%). The 12 atypical enterococcal species (5%) that showed aberrant sugar reactions in conventional phenotyping were confirmed as  E. faecalis, E. faecium  and  E. casseliflavus  respectively by WCP fingerprinting. The antimicrobial susceptibility testing depicted the emergence of high-level aminoglycoside and beta-lactam resistance among different species apart from intrinsic vancomycin resistance by some species, while all the species tested were susceptible for linezolid and teicoplanin.E. gallinarumE. aviumE. raffinosusE. hiraeE. mundtiiE. casseliflavusE. duransE. faecalis, E. faeciumE. casseliflavusConclusion Our study reveals the emergence of multi-drug resistance among unusual species of enterococci posing a serious therapeutic challenge. Precise identification of enterococci to species level enables us to access the species-specific antimicrobial resistance characteristics, apart from knowing the epidemiological pattern and their clinical significance in human infections.
Conclusion
Our study reveals the emergence of multi-drug resistance among unusual species of enterococci posing a serious therapeutic challenge. Precise identification of enterococci to species level enables us to access the species-specific antimicrobial resistance characteristics, apart from knowing the epidemiological pattern and their clinical significance in human infections.

Background Enterococci, generally regarded as normal flora of gastrointestinal and genitourinary tract of humans, have emerged as the etiogen of several nosocomial as well community-acquired infections since last two decades. Globally, many studies have revealed that enterococci tend to be one of the leading causes of several nosocomial infections, with the emergence and spread of multi drug resistance among isolates [ 1 - 3 ]. Since the inception of separate genus Enterococcus, there are 23 species of enterococci with clinical significance to date [ 4 ], of which  Enterococcus faecalis  and  Enterococcus faecium  accounts up to 90% of clinical isolates belonging to this genus [ 1 ]. Nevertheless, the incidence of other species of enterococci from clinical sources shows an alarming increase with the properties of intrinsic resistance to several antibiotics including beta-lactams and glycopeptides [ 5 , 6 ]. But the incidence of non-faecalis and non-faecium enterococci is underestimated because of frequent misidentification. On several instances only one phenotypic character differentiates one species from another, and to further complicate some strains of enterococci do not posses the exact phenotypic character of the type strains, and there comes confusion over their exact taxonomic status [ 7 ]. Thus proper identification of enterococci to species level is quintessential for management and prevention of these bacteria in any health care facility. Many studies focus on the two most common species  E. faecalis  and  E. faecium , and only few reports or studies of non-faecalis and non-faecium enterococci are prevalent [ 5 , 6 ]. Hence the aim of our study was to check the prevalence of unusual and atypical species of enterococci causing human infections, in a tertiary care hospital in South India over a time period.
Background
Enterococci, generally regarded as normal flora of gastrointestinal and genitourinary tract of humans, have emerged as the etiogen of several nosocomial as well community-acquired infections since last two decades. Globally, many studies have revealed that enterococci tend to be one of the leading causes of several nosocomial infections, with the emergence and spread of multi drug resistance among isolates [ 1 - 3 ]. Since the inception of separate genus Enterococcus, there are 23 species of enterococci with clinical significance to date [ 4 ], of which  Enterococcus faecalis  and  Enterococcus faecium  accounts up to 90% of clinical isolates belonging to this genus [ 1 ]. Nevertheless, the incidence of other species of enterococci from clinical sources shows an alarming increase with the properties of intrinsic resistance to several antibiotics including beta-lactams and glycopeptides [ 5 , 6 ]. But the incidence of non-faecalis and non-faecium enterococci is underestimated because of frequent misidentification. On several instances only one phenotypic character differentiates one species from another, and to further complicate some strains of enterococci do not posses the exact phenotypic character of the type strains, and there comes confusion over their exact taxonomic status [ 7 ]. Thus proper identification of enterococci to species level is quintessential for management and prevention of these bacteria in any health care facility. Many studies focus on the two most common species  E. faecalis  and  E. faecium , and only few reports or studies of non-faecalis and non-faecium enterococci are prevalent [ 5 , 6 ]. Hence the aim of our study was to check the prevalence of unusual and atypical species of enterococci causing human infections, in a tertiary care hospital in South India over a time period.Enterococcus faecalisEnterococcus faeciumE. faecalisE. faeciumMethods i. Bacterial isolates and conventional phenotypic characterization of enterococci The study was conducted in a 900-bedded tertiary care hospital at Pondicherry, South India from July 2001 to June 2003. Isolates of enterococci were collected over the time period from various clinical specimens such as blood, urine, wound swabs and pus (surgical and non-surgical), catheters, ascitic fluid, synovial fluid, by plating them on 5% Sheep Blood agar and Mac-conkey agar, as well on Bile esculin azide agar (Hi-media, Mumbai, India) as per nature of the specimen. Extensive phenotypic and physiological characterization was carried out by the conventional tests devised by Facklam et al [ 3 , 8 ]. Carbohydrate fermentation tests were performed using 1% sugar discs in Brain heart infusion (BHI) broth with Andrade's indicator (Hi-media, Mumbai, India) as per manufacturer's instructions. The following sugars were tested for fermentation by isolates using commercial discs: mannitol, sorbitol, inulin, arabinose, melibiose, sucrose, raffinose, trehalose, lactose, glycerol, salicin, maltose, adonitol, and xylose, while sorbose and ribose were added to a final concentration of 1% to the broth base directly after sterilization (due to non-availability of discs). Group D antigen was detected using a commercial latex agglutination kit (The Binding site limited, Birmingham, B29 6AT) as per manufacturer's™ instructions. ii. Antimicrobial susceptibility testing Antibiotic susceptibility testing of the clinical isolates along with the quality control strains were performed using BHI agar instead of Muller Hinton agar by disk diffusion method (for the antibiotics: penicillin [10 units], ampicillin [10 μg], gentamicin-high content [120 μg], streptomycin-high content [300 μg], ciprofloxacin [5 μg], nitrofurantoin-for urinary isolates only [300 μg], vancomycin [30 μg], teicoplanin [30 μg] and linezolid [30 μg]), standard agar dilution (for the antibiotics mentioned in Table- 1 ) and agar screening methods (for vancomycin and high-level aminoglycoside resistance) and interpreted as per NCCLS guidelines [ 9 ]. Production of β-lactamase was determined by using nitrocefin discs (BBL Microsystems) as per manufacturer's™ instructions. Table 1 Analysis of MIC ranges of unusual species of enterococci. Species tested (no.of.isolates) Antibiotic Tested No. of isolates at specified MIC, in μg/mL Susc a ,% 2 4 8 16 32 ≥ 64 E. avium  (10) Pen. 9 9 9 9 7 6 10 Amp. 9 6 6 6 6 0 40 Van. 4 2 0 0 0 0 100 Te. 0 0 0 0 0 0 100 Va.Scr. NA NA NA NA NA NA 100 HLGm. NA NA NA NA NA NA 10 HLStr. NA NA NA NA NA NA 50 E. casseliflavus  (3) Pen. 0 0 0 0 0 0 100 Amp. 0 0 0 0 0 0 100 Van. 3 3 0 0 0 0 NA Te. 0 0 0 0 0 0 100 Va.Scr. NA NA NA NA NA NA 66.6 HLGm. NA NA NA NA NA NA 100 HLStr. NA NA NA NA NA NA 100 E. durans  (2) Pen. 2 1 1 1 0 0 50 Amp. 1 1 1 1 0 0 50 Van. 2 1 0 0 0 0 100 Te. 1 0 0 0 0 0 100 Va.Scr. NA NA NA NA NA NA 50 HLGm. NA NA NA NA NA NA 50 HLStr. NA NA NA NA NA NA 0 E. gallinarum  (15) Pen. 9 9 8 8 8 8 46.6 Amp. 8 8 8 8 7 6 46.6 Van. 13 9 2 0 0 0 NA Te. 0 0 0 0 0 0 100 Va.Scr. NA NA NA NA NA NA 53.3 HLGm. NA NA NA NA NA NA 46.6 HLStr. NA NA NA NA NA NA 66.6 E. hirae  (6) Pen. 3 3 2 2 0 0 66.6 Amp. 6 2 2 2 0 0 66.6 Van. 0 0 0 0 0 0 100 Te. 0 0 0 0 0 0 100 Va.Scr. NA NA NA NA NA NA 100 HLGm. NA NA NA NA NA NA 100 HLStr. NA NA NA NA NA NA 100 E. mundtii  (4) Pen. 1 1 1 0 0 0 100 Amp. 0 0 0 0 0 0 100 Van. 2 2 0 0 0 0 100 Te. 0 0 0 0 0 0 100 Va.Scr. NA NA NA NA NA NA 50 HLGm. NA NA NA NA NA NA 100 HLStr. NA NA NA NA NA NA 50 E. raffinosus  (6) Pen. 4 4 4 4 4 4 33.3 Amp. 4 4 4 4 4 0 33.3 Van. 0 0 0 0 0 0 100 Te. 0 0 0 0 0 0 100 Va.Scr. NA NA NA NA NA NA 100 HLGm. NA NA NA NA NA NA 66.6 HLStr. NA NA NA NA NA NA 66.6 NOTE: a  Interpretations based on NCCLS guidelines, NA-not applicable; Susc- Susceptiblity, Pen-Penicillin; Amp-Ampicillin; Van-Vancomycin; Te-Teicoplanin, Va. Scr- Vancomycin resistance (6 μg/mL) agar screening, HLGm- High-level gentamicin resistance (500 μg/mL) agar screening, HLStr- High-level streptomycin resistance (2000 μg/mL) agar screening, iii. Molecular phenotyping of enterococci Whole cell protein (WCP) analysis of the enterococcal isolates, including atypical biochemical variants of enterococci and the reference/type strains of enterococci (a kind gift from Dr. Richard.R.Facklam, CDC, Atlanta, GA. USA) were done using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously with minor modifications [ 10 , 11 ] for species identification, as well confirmation of species identities of atypical strains. Briefly, enterococcal test strains were grown for 18 hours at 37°C on Trypticase soy agar with 5% sheep blood. The samples were prepared by removing the bacterial growth from the surface of agar plate carefully with a sterile disposable loop, and suspended in 5 ml of sterile saline solution in order to obtain a turbidity equal to that of No.8 MacFarland density standard, centrifuged, and resuspended in 0.5 ml of an aqueous lysozyme solution (10 mg/ml). The suspensions were incubated in a water bath preset at 37°C for 2 hours. The WCP extracts were obtained by mixing one part of whole cell extract to one part of sample loading buffer, and boiled for 5 minutes and separated by SDS-PAGE along with a broad range molecular weight marker (New England Biolabs Inc.,) as per standard procedure [ 11 ]. The SDS-PAGE was performed using 5% stacking gel and 10% separating gel at a constant current of 20 mA using a mini-gel electrophoresis system (Bangalore Genei, India) and stained with Coomassie brilliant blue. Visual comparisons of the gels were made, and documentation done using a Gel doc system (Vilber loubert, France) for further analysis. The gel images were analyzed and dendrogram constructed using appropriate software (Bionumerics, version 2.5, Applied Maths, Kortrijik, Belgium) for validating the taxonomy of the enterococcal species studied.
Methods
i. Bacterial isolates and conventional phenotypic characterization of enterococci The study was conducted in a 900-bedded tertiary care hospital at Pondicherry, South India from July 2001 to June 2003. Isolates of enterococci were collected over the time period from various clinical specimens such as blood, urine, wound swabs and pus (surgical and non-surgical), catheters, ascitic fluid, synovial fluid, by plating them on 5% Sheep Blood agar and Mac-conkey agar, as well on Bile esculin azide agar (Hi-media, Mumbai, India) as per nature of the specimen. Extensive phenotypic and physiological characterization was carried out by the conventional tests devised by Facklam et al [ 3 , 8 ]. Carbohydrate fermentation tests were performed using 1% sugar discs in Brain heart infusion (BHI) broth with Andrade's indicator (Hi-media, Mumbai, India) as per manufacturer's instructions. The following sugars were tested for fermentation by isolates using commercial discs: mannitol, sorbitol, inulin, arabinose, melibiose, sucrose, raffinose, trehalose, lactose, glycerol, salicin, maltose, adonitol, and xylose, while sorbose and ribose were added to a final concentration of 1% to the broth base directly after sterilization (due to non-availability of discs). Group D antigen was detected using a commercial latex agglutination kit (The Binding site limited, Birmingham, B29 6AT) as per manufacturer's™ instructions.
i. Bacterial isolates and conventional phenotypic characterization of enterococci
The study was conducted in a 900-bedded tertiary care hospital at Pondicherry, South India from July 2001 to June 2003. Isolates of enterococci were collected over the time period from various clinical specimens such as blood, urine, wound swabs and pus (surgical and non-surgical), catheters, ascitic fluid, synovial fluid, by plating them on 5% Sheep Blood agar and Mac-conkey agar, as well on Bile esculin azide agar (Hi-media, Mumbai, India) as per nature of the specimen. Extensive phenotypic and physiological characterization was carried out by the conventional tests devised by Facklam et al [ 3 , 8 ]. Carbohydrate fermentation tests were performed using 1% sugar discs in Brain heart infusion (BHI) broth with Andrade's indicator (Hi-media, Mumbai, India) as per manufacturer's instructions. The following sugars were tested for fermentation by isolates using commercial discs: mannitol, sorbitol, inulin, arabinose, melibiose, sucrose, raffinose, trehalose, lactose, glycerol, salicin, maltose, adonitol, and xylose, while sorbose and ribose were added to a final concentration of 1% to the broth base directly after sterilization (due to non-availability of discs). Group D antigen was detected using a commercial latex agglutination kit (The Binding site limited, Birmingham, B29 6AT) as per manufacturer's™ instructions.ii. Antimicrobial susceptibility testing Antibiotic susceptibility testing of the clinical isolates along with the quality control strains were performed using BHI agar instead of Muller Hinton agar by disk diffusion method (for the antibiotics: penicillin [10 units], ampicillin [10 μg], gentamicin-high content [120 μg], streptomycin-high content [300 μg], ciprofloxacin [5 μg], nitrofurantoin-for urinary isolates only [300 μg], vancomycin [30 μg], teicoplanin [30 μg] and linezolid [30 μg]), standard agar dilution (for the antibiotics mentioned in Table- 1 ) and agar screening methods (for vancomycin and high-level aminoglycoside resistance) and interpreted as per NCCLS guidelines [ 9 ]. Production of β-lactamase was determined by using nitrocefin discs (BBL Microsystems) as per manufacturer's™ instructions. Table 1 Analysis of MIC ranges of unusual species of enterococci. Species tested (no.of.isolates) Antibiotic Tested No. of isolates at specified MIC, in μg/mL Susc a ,% 2 4 8 16 32 ≥ 64 E. avium  (10) Pen. 9 9 9 9 7 6 10 Amp. 9 6 6 6 6 0 40 Van. 4 2 0 0 0 0 100 Te. 0 0 0 0 0 0 100 Va.Scr. NA NA NA NA NA NA 100 HLGm. NA NA NA NA NA NA 10 HLStr. NA NA NA NA NA NA 50 E. casseliflavus  (3) Pen. 0 0 0 0 0 0 100 Amp. 0 0 0 0 0 0 100 Van. 3 3 0 0 0 0 NA Te. 0 0 0 0 0 0 100 Va.Scr. NA NA NA NA NA NA 66.6 HLGm. NA NA NA NA NA NA 100 HLStr. NA NA NA NA NA NA 100 E. durans  (2) Pen. 2 1 1 1 0 0 50 Amp. 1 1 1 1 0 0 50 Van. 2 1 0 0 0 0 100 Te. 1 0 0 0 0 0 100 Va.Scr. NA NA NA NA NA NA 50 HLGm. NA NA NA NA NA NA 50 HLStr. NA NA NA NA NA NA 0 E. gallinarum  (15) Pen. 9 9 8 8 8 8 46.6 Amp. 8 8 8 8 7 6 46.6 Van. 13 9 2 0 0 0 NA Te. 0 0 0 0 0 0 100 Va.Scr. NA NA NA NA NA NA 53.3 HLGm. NA NA NA NA NA NA 46.6 HLStr. NA NA NA NA NA NA 66.6 E. hirae  (6) Pen. 3 3 2 2 0 0 66.6 Amp. 6 2 2 2 0 0 66.6 Van. 0 0 0 0 0 0 100 Te. 0 0 0 0 0 0 100 Va.Scr. NA NA NA NA NA NA 100 HLGm. NA NA NA NA NA NA 100 HLStr. NA NA NA NA NA NA 100 E. mundtii  (4) Pen. 1 1 1 0 0 0 100 Amp. 0 0 0 0 0 0 100 Van. 2 2 0 0 0 0 100 Te. 0 0 0 0 0 0 100 Va.Scr. NA NA NA NA NA NA 50 HLGm. NA NA NA NA NA NA 100 HLStr. NA NA NA NA NA NA 50 E. raffinosus  (6) Pen. 4 4 4 4 4 4 33.3 Amp. 4 4 4 4 4 0 33.3 Van. 0 0 0 0 0 0 100 Te. 0 0 0 0 0 0 100 Va.Scr. NA NA NA NA NA NA 100 HLGm. NA NA NA NA NA NA 66.6 HLStr. NA NA NA NA NA NA 66.6 NOTE: a  Interpretations based on NCCLS guidelines, NA-not applicable; Susc- Susceptiblity, Pen-Penicillin; Amp-Ampicillin; Van-Vancomycin; Te-Teicoplanin, Va. Scr- Vancomycin resistance (6 μg/mL) agar screening, HLGm- High-level gentamicin resistance (500 μg/mL) agar screening, HLStr- High-level streptomycin resistance (2000 μg/mL) agar screening,
ii. Antimicrobial susceptibility testing
Antibiotic susceptibility testing of the clinical isolates along with the quality control strains were performed using BHI agar instead of Muller Hinton agar by disk diffusion method (for the antibiotics: penicillin [10 units], ampicillin [10 μg], gentamicin-high content [120 μg], streptomycin-high content [300 μg], ciprofloxacin [5 μg], nitrofurantoin-for urinary isolates only [300 μg], vancomycin [30 μg], teicoplanin [30 μg] and linezolid [30 μg]), standard agar dilution (for the antibiotics mentioned in Table- 1 ) and agar screening methods (for vancomycin and high-level aminoglycoside resistance) and interpreted as per NCCLS guidelines [ 9 ]. Production of β-lactamase was determined by using nitrocefin discs (BBL Microsystems) as per manufacturer's™ instructions.Analysis of MIC ranges of unusual species of enterococci.E. aviumE. casseliflavusE. duransE. gallinarumE. hiraeE. mundtiiE. raffinosusNOTE:a  Interpretations based on NCCLS guidelines,NA-not applicable; Susc- Susceptiblity,Pen-Penicillin; Amp-Ampicillin; Van-Vancomycin; Te-Teicoplanin,Va. Scr- Vancomycin resistance (6 μg/mL) agar screening,HLGm- High-level gentamicin resistance (500 μg/mL) agar screening,HLStr- High-level streptomycin resistance (2000 μg/mL) agar screening,iii. Molecular phenotyping of enterococci Whole cell protein (WCP) analysis of the enterococcal isolates, including atypical biochemical variants of enterococci and the reference/type strains of enterococci (a kind gift from Dr. Richard.R.Facklam, CDC, Atlanta, GA. USA) were done using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously with minor modifications [ 10 , 11 ] for species identification, as well confirmation of species identities of atypical strains. Briefly, enterococcal test strains were grown for 18 hours at 37°C on Trypticase soy agar with 5% sheep blood. The samples were prepared by removing the bacterial growth from the surface of agar plate carefully with a sterile disposable loop, and suspended in 5 ml of sterile saline solution in order to obtain a turbidity equal to that of No.8 MacFarland density standard, centrifuged, and resuspended in 0.5 ml of an aqueous lysozyme solution (10 mg/ml). The suspensions were incubated in a water bath preset at 37°C for 2 hours. The WCP extracts were obtained by mixing one part of whole cell extract to one part of sample loading buffer, and boiled for 5 minutes and separated by SDS-PAGE along with a broad range molecular weight marker (New England Biolabs Inc.,) as per standard procedure [ 11 ]. The SDS-PAGE was performed using 5% stacking gel and 10% separating gel at a constant current of 20 mA using a mini-gel electrophoresis system (Bangalore Genei, India) and stained with Coomassie brilliant blue. Visual comparisons of the gels were made, and documentation done using a Gel doc system (Vilber loubert, France) for further analysis. The gel images were analyzed and dendrogram constructed using appropriate software (Bionumerics, version 2.5, Applied Maths, Kortrijik, Belgium) for validating the taxonomy of the enterococcal species studied.
iii. Molecular phenotyping of enterococci
Whole cell protein (WCP) analysis of the enterococcal isolates, including atypical biochemical variants of enterococci and the reference/type strains of enterococci (a kind gift from Dr. Richard.R.Facklam, CDC, Atlanta, GA. USA) were done using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described previously with minor modifications [ 10 , 11 ] for species identification, as well confirmation of species identities of atypical strains. Briefly, enterococcal test strains were grown for 18 hours at 37°C on Trypticase soy agar with 5% sheep blood. The samples were prepared by removing the bacterial growth from the surface of agar plate carefully with a sterile disposable loop, and suspended in 5 ml of sterile saline solution in order to obtain a turbidity equal to that of No.8 MacFarland density standard, centrifuged, and resuspended in 0.5 ml of an aqueous lysozyme solution (10 mg/ml). The suspensions were incubated in a water bath preset at 37°C for 2 hours. The WCP extracts were obtained by mixing one part of whole cell extract to one part of sample loading buffer, and boiled for 5 minutes and separated by SDS-PAGE along with a broad range molecular weight marker (New England Biolabs Inc.,) as per standard procedure [ 11 ]. The SDS-PAGE was performed using 5% stacking gel and 10% separating gel at a constant current of 20 mA using a mini-gel electrophoresis system (Bangalore Genei, India) and stained with Coomassie brilliant blue. Visual comparisons of the gels were made, and documentation done using a Gel doc system (Vilber loubert, France) for further analysis. The gel images were analyzed and dendrogram constructed using appropriate software (Bionumerics, version 2.5, Applied Maths, Kortrijik, Belgium) for validating the taxonomy of the enterococcal species studied.Results Conventional and Molecular phenotyping of enterococci We isolated a total of 242 enterococci during our 2-year study period from different clinical samples. The biochemical phenotyping results revealed 46 isolates (19%) belonging to 7 different unusual species of enterococci (excluding  E. faecalis  and  E. faecium -data not shown) which included 15  E. gallinarum  (6.2%), 10  E. avium  (4.1%), 6  E. raffinosus  (2.5%), 6  E. hirae  (2.5%), 4  E. mundtii  (1.7%), 3  E. casseliflavus  (1.2%) and 2  E. durans  (0.8%). The distribution by site of isolation for the 46 unusual enterococcal species included 30 isolates- 12  E. gallinarum , 6  E. avium , 3 each of  E. hirae ,  E. casseliflavus  and  E. raffinosus , 2  E. mundtii  and 1  E. durans  (65.2%) from bloodstream, 6 isolates- 3  E. raffinosus , 2  E. avium  and 1  E. mundtii  (13 %) from surgical and non-surgical wound swabs, 10 isolates- 3 each of  E. hirae  and  E. gallinarum , 2  E. avium  and 1 each of  E. durans  and  E. mundtii  (21.8%) from miscellaneous sites, including muscle tissues sent for anaerobic culture, catheter tips, peritoneal fluid, ear swab and urine. Of the 46 persons from whom unusual enterococci were obtained, 56.5% were males and 43.5% were females including newborn/neonates. The infections were polymicrobial in 6 (13%) of the 46 cases from which unusual enterococci were isolated, including 2 (6.7%) of 30 bloodstream infections. The 12 atypical enterococcal strains (5%) showing aberrant sugar reactions included 6 mannitol negative variant  E. faecalis  like species, 1 arginine negative variant  E. faecalis  like species, 3 mannitol negative variant  E. faecium  like species and, 2 arginine negative variant  E. casseliflavus  like species. The WCP analysis by SDS-PAGE confirmed the species identities of seven different species. Atypical strains showed a similar banding pattern like the reference strains from CDC ( E. faecalis  SS-1273,  E. faecium  SS-1274,  E. casseliflavus  SS-1229) except for minor quantitative differences, with no qualitative difference. The computational analysis of the WCP gel images of atypical strains were performed by Dice coefficient, and the dendrogram constructed using unweighted pair group method using arithmetic averages (UPGMA) as shown in Figure- 1 , and validated their exact taxonomic status as  E. faecalis, E. faecium  and  E. casseliflavus  respectively. The 2 (atypical) isolates of arginine negative variant  E. casseliflavus  like species after taxonomic validation were included as an unusual species of enterococci accounting to 3  E. casseliflavus  isolated overall. Figure 1 Cluster analysis of atypical strains of  Enterococci  using Dice coefficient and UPGMA method (Bionumerics, Applied Maths, Belgium). Note : SS- Designation of CDC standard strains,  E. porcinosus  is currently designated as  E. villorum ,  E. pseudoav.- E. pseudoavium ;  E. malodorat.- E. malodoratus ,  E. casselifla.- E. casseliflavus , MNV- Mannitol negative variant; ANV- Arginine negative variant. Antimicrobial susceptibility testing The antimicrobial susceptibilities of the isolates given in Table- 1  depict the ranges of MICs for various antimicrobial agents tested by standard agar dilution, and agar screening methods. The  E. gallinarum  and  E. casseliflavus  isolates showed reduced susceptibility to lesser concentrations of vancomycin ranging 2–8 μg/ml. Other species were highly susceptible for vancomycin and teicoplanin except one isolate of  E. durans . High-level aminoglycoside resistance for gentamicin and streptomycin was found absent only in  E. casseliflavus  and  E. hirae , while other species exhibited variable susceptibilities ranging 0 – 66.7% for either aminoglycoside tested. The disk diffusion testing showed 100% susceptibility for linezolid and teicoplanin by all isolates tested, while  E. casseliflavus  and  E. mundtii  showed 100% susceptibility for penicillin and ampicillin. Only 37% of unusual enterococcal isolates were susceptible to ciprofloxacin, with resistance exhibited by 9  E. avium  (n = 10), 2  E. durans  (n = 2), 11  E. gallinarum  (n = 15), 2  E. hirae  (n = 6), 2  E. mundtii  (n = 4) and, 3  E. raffinosus  (n = 6). Only  E. casseliflavus  (n = 3) exhibited 100% susceptibility to ciprofloxacin. None of the 46 isolates was positive for β-lactamase, but resistance for β-lactam agents were prevalent variably among different species. The results of MICs for penicillin, ampicillin, high-level gentamicin and high-level streptomycin resistance were in accordance with the disk diffusion testing results except for vancomycin. Disk diffusion testing showed vancomycin resistance for 6 isolates (1  E. durans , 2  E. mundtii , 3  E. gallinarum ), but the agar screening method exhibited vancomycin resistance for 11 isolates (2  E. mundtii , 1  E. casseliflavus , 1  E. durans , 7  E. gallinarum )(including 5 isolates-4  E. gallinarum  and 1  E. casseliflavus , which showed susceptibility to vancomycin by the disk diffusion method).
Results
Conventional and Molecular phenotyping of enterococci We isolated a total of 242 enterococci during our 2-year study period from different clinical samples. The biochemical phenotyping results revealed 46 isolates (19%) belonging to 7 different unusual species of enterococci (excluding  E. faecalis  and  E. faecium -data not shown) which included 15  E. gallinarum  (6.2%), 10  E. avium  (4.1%), 6  E. raffinosus  (2.5%), 6  E. hirae  (2.5%), 4  E. mundtii  (1.7%), 3  E. casseliflavus  (1.2%) and 2  E. durans  (0.8%). The distribution by site of isolation for the 46 unusual enterococcal species included 30 isolates- 12  E. gallinarum , 6  E. avium , 3 each of  E. hirae ,  E. casseliflavus  and  E. raffinosus , 2  E. mundtii  and 1  E. durans  (65.2%) from bloodstream, 6 isolates- 3  E. raffinosus , 2  E. avium  and 1  E. mundtii  (13 %) from surgical and non-surgical wound swabs, 10 isolates- 3 each of  E. hirae  and  E. gallinarum , 2  E. avium  and 1 each of  E. durans  and  E. mundtii  (21.8%) from miscellaneous sites, including muscle tissues sent for anaerobic culture, catheter tips, peritoneal fluid, ear swab and urine. Of the 46 persons from whom unusual enterococci were obtained, 56.5% were males and 43.5% were females including newborn/neonates. The infections were polymicrobial in 6 (13%) of the 46 cases from which unusual enterococci were isolated, including 2 (6.7%) of 30 bloodstream infections. The 12 atypical enterococcal strains (5%) showing aberrant sugar reactions included 6 mannitol negative variant  E. faecalis  like species, 1 arginine negative variant  E. faecalis  like species, 3 mannitol negative variant  E. faecium  like species and, 2 arginine negative variant  E. casseliflavus  like species. The WCP analysis by SDS-PAGE confirmed the species identities of seven different species. Atypical strains showed a similar banding pattern like the reference strains from CDC ( E. faecalis  SS-1273,  E. faecium  SS-1274,  E. casseliflavus  SS-1229) except for minor quantitative differences, with no qualitative difference. The computational analysis of the WCP gel images of atypical strains were performed by Dice coefficient, and the dendrogram constructed using unweighted pair group method using arithmetic averages (UPGMA) as shown in Figure- 1 , and validated their exact taxonomic status as  E. faecalis, E. faecium  and  E. casseliflavus  respectively. The 2 (atypical) isolates of arginine negative variant  E. casseliflavus  like species after taxonomic validation were included as an unusual species of enterococci accounting to 3  E. casseliflavus  isolated overall. Figure 1 Cluster analysis of atypical strains of  Enterococci  using Dice coefficient and UPGMA method (Bionumerics, Applied Maths, Belgium). Note : SS- Designation of CDC standard strains,  E. porcinosus  is currently designated as  E. villorum ,  E. pseudoav.- E. pseudoavium ;  E. malodorat.- E. malodoratus ,  E. casselifla.- E. casseliflavus , MNV- Mannitol negative variant; ANV- Arginine negative variant.
Conventional and Molecular phenotyping of enterococci
We isolated a total of 242 enterococci during our 2-year study period from different clinical samples. The biochemical phenotyping results revealed 46 isolates (19%) belonging to 7 different unusual species of enterococci (excluding  E. faecalis  and  E. faecium -data not shown) which included 15  E. gallinarum  (6.2%), 10  E. avium  (4.1%), 6  E. raffinosus  (2.5%), 6  E. hirae  (2.5%), 4  E. mundtii  (1.7%), 3  E. casseliflavus  (1.2%) and 2  E. durans  (0.8%). The distribution by site of isolation for the 46 unusual enterococcal species included 30 isolates- 12  E. gallinarum , 6  E. avium , 3 each of  E. hirae ,  E. casseliflavus  and  E. raffinosus , 2  E. mundtii  and 1  E. durans  (65.2%) from bloodstream, 6 isolates- 3  E. raffinosus , 2  E. avium  and 1  E. mundtii  (13 %) from surgical and non-surgical wound swabs, 10 isolates- 3 each of  E. hirae  and  E. gallinarum , 2  E. avium  and 1 each of  E. durans  and  E. mundtii  (21.8%) from miscellaneous sites, including muscle tissues sent for anaerobic culture, catheter tips, peritoneal fluid, ear swab and urine. Of the 46 persons from whom unusual enterococci were obtained, 56.5% were males and 43.5% were females including newborn/neonates. The infections were polymicrobial in 6 (13%) of the 46 cases from which unusual enterococci were isolated, including 2 (6.7%) of 30 bloodstream infections. The 12 atypical enterococcal strains (5%) showing aberrant sugar reactions included 6 mannitol negative variant  E. faecalis  like species, 1 arginine negative variant  E. faecalis  like species, 3 mannitol negative variant  E. faecium  like species and, 2 arginine negative variant  E. casseliflavus  like species. The WCP analysis by SDS-PAGE confirmed the species identities of seven different species. Atypical strains showed a similar banding pattern like the reference strains from CDC ( E. faecalis  SS-1273,  E. faecium  SS-1274,  E. casseliflavus  SS-1229) except for minor quantitative differences, with no qualitative difference. The computational analysis of the WCP gel images of atypical strains were performed by Dice coefficient, and the dendrogram constructed using unweighted pair group method using arithmetic averages (UPGMA) as shown in Figure- 1 , and validated their exact taxonomic status as  E. faecalis, E. faecium  and  E. casseliflavus  respectively. The 2 (atypical) isolates of arginine negative variant  E. casseliflavus  like species after taxonomic validation were included as an unusual species of enterococci accounting to 3  E. casseliflavus  isolated overall.E. faecalisE. faeciumE. gallinarumE. aviumE. raffinosusE. hiraeE. mundtiiE. casseliflavusE. duransE. gallinarumE. aviumE. hiraeE. casseliflavusE. raffinosusE. mundtiiE. duransE. raffinosusE. aviumE. mundtiiE. hiraeE. gallinarumE. aviumE. duransE. mundtiiE. faecalisE. faecalisE. faeciumE. casseliflavusE. faecalisE. faeciumE. casseliflavusE. faecalis, E. faeciumE. casseliflavusE. casseliflavusE. casseliflavusAntimicrobial susceptibility testing The antimicrobial susceptibilities of the isolates given in Table- 1  depict the ranges of MICs for various antimicrobial agents tested by standard agar dilution, and agar screening methods. The  E. gallinarum  and  E. casseliflavus  isolates showed reduced susceptibility to lesser concentrations of vancomycin ranging 2–8 μg/ml. Other species were highly susceptible for vancomycin and teicoplanin except one isolate of  E. durans . High-level aminoglycoside resistance for gentamicin and streptomycin was found absent only in  E. casseliflavus  and  E. hirae , while other species exhibited variable susceptibilities ranging 0 – 66.7% for either aminoglycoside tested. The disk diffusion testing showed 100% susceptibility for linezolid and teicoplanin by all isolates tested, while  E. casseliflavus  and  E. mundtii  showed 100% susceptibility for penicillin and ampicillin. Only 37% of unusual enterococcal isolates were susceptible to ciprofloxacin, with resistance exhibited by 9  E. avium  (n = 10), 2  E. durans  (n = 2), 11  E. gallinarum  (n = 15), 2  E. hirae  (n = 6), 2  E. mundtii  (n = 4) and, 3  E. raffinosus  (n = 6). Only  E. casseliflavus  (n = 3) exhibited 100% susceptibility to ciprofloxacin. None of the 46 isolates was positive for β-lactamase, but resistance for β-lactam agents were prevalent variably among different species. The results of MICs for penicillin, ampicillin, high-level gentamicin and high-level streptomycin resistance were in accordance with the disk diffusion testing results except for vancomycin. Disk diffusion testing showed vancomycin resistance for 6 isolates (1  E. durans , 2  E. mundtii , 3  E. gallinarum ), but the agar screening method exhibited vancomycin resistance for 11 isolates (2  E. mundtii , 1  E. casseliflavus , 1  E. durans , 7  E. gallinarum )(including 5 isolates-4  E. gallinarum  and 1  E. casseliflavus , which showed susceptibility to vancomycin by the disk diffusion method).
Antimicrobial susceptibility testing
The antimicrobial susceptibilities of the isolates given in Table- 1  depict the ranges of MICs for various antimicrobial agents tested by standard agar dilution, and agar screening methods. The  E. gallinarum  and  E. casseliflavus  isolates showed reduced susceptibility to lesser concentrations of vancomycin ranging 2–8 μg/ml. Other species were highly susceptible for vancomycin and teicoplanin except one isolate of  E. durans . High-level aminoglycoside resistance for gentamicin and streptomycin was found absent only in  E. casseliflavus  and  E. hirae , while other species exhibited variable susceptibilities ranging 0 – 66.7% for either aminoglycoside tested. The disk diffusion testing showed 100% susceptibility for linezolid and teicoplanin by all isolates tested, while  E. casseliflavus  and  E. mundtii  showed 100% susceptibility for penicillin and ampicillin. Only 37% of unusual enterococcal isolates were susceptible to ciprofloxacin, with resistance exhibited by 9  E. avium  (n = 10), 2  E. durans  (n = 2), 11  E. gallinarum  (n = 15), 2  E. hirae  (n = 6), 2  E. mundtii  (n = 4) and, 3  E. raffinosus  (n = 6). Only  E. casseliflavus  (n = 3) exhibited 100% susceptibility to ciprofloxacin. None of the 46 isolates was positive for β-lactamase, but resistance for β-lactam agents were prevalent variably among different species. The results of MICs for penicillin, ampicillin, high-level gentamicin and high-level streptomycin resistance were in accordance with the disk diffusion testing results except for vancomycin. Disk diffusion testing showed vancomycin resistance for 6 isolates (1  E. durans , 2  E. mundtii , 3  E. gallinarum ), but the agar screening method exhibited vancomycin resistance for 11 isolates (2  E. mundtii , 1  E. casseliflavus , 1  E. durans , 7  E. gallinarum )(including 5 isolates-4  E. gallinarum  and 1  E. casseliflavus , which showed susceptibility to vancomycin by the disk diffusion method).E. gallinarumE. casseliflavusE. duransE. casseliflavusE. hiraeE. casseliflavusE. mundtiiE. aviumE. duransE. gallinarumE. hiraeE. mundtiiE. raffinosusE. casseliflavusE. duransE. mundtiiE. gallinarumE. mundtiiE. casseliflavusE. duransE. gallinarumE. gallinarumE. casseliflavusDiscussion Our study reveals that the prevalence of unusual species of enterococci as 19% in our clinical setup in South India. Many studies and reviews show the prevalence of non-faecalis and non-faecium enterococci as 2–10% [ 3 , 6 , 12 ]. Previous studies from India have reported  E. faecalis  and  E. faecium  as the only prevalent species [ 13 - 16 ], which may not reflect the true incidence rate. From our perspective the real incidence tends to be higher which in part can be explained as, misidentification of species due to exhibition of aberrant sugar reactions by some enterococci or, due to lack of application of the complete range of tests to identify non-faecalis and non-faecium enterococci [ 7 , 17 ]. The prevalence rate (19%) of our study was partly in accordance with another Indian study [ 18 ] showing 14.8% (excluding  E. faecalis  and  E. faecium ) prevalence of unusual species of enterococci from catheterized patients with urinary tract infections.  E. mundtii  and  E. durans  were not reported in their study, whose prevalence was 1.7% and 0.8% respectively in our study.  E. gallinarum  (6.2%) and  E. avium  (4.1%) were the most commonly identified species, which markedly differs in isolation rate (0.3–1.2%) from other studies [ 6 , 19 , 20 ]. The incidence of infections caused by unusual enterococcal species is of serious concern, since 43.5% of the isolates were from cases of septicemia without endocarditis. Apart from septicemia, the unusual species of enterococci were isolated frequently from cases of urinary tract infections, surgical and non-surgical wound infections and peritonitis. Most of the patients with the bloodstream infections had a peripheral or central catheter. Further, only 13% of enterococcal infections were polymicrobial, with majority from non-bloodstream isolates that underscores the clinical significance of these unusual enterococcal species. Although the unusual species of enterococci were isolated at regular intervals throughout our study period, we could find clustering of specific species during a specific time period from specific units/wards. Interestingly, 10 among the 15 isolates of  E. gallinarum  isolated during our study period were from pediatrics unit, while 7 of the 10 isolates exhibiting a similar antibiotype were isolated from the same ward within a span of 2 months. The remaining 3 of the 10  E. gallinarum  were isolated from the same ward in the preceding 3 months, one of which showed an antibiotype similar to the cluster of 7 isolates. The same was the case of 3  E. casseliflavus  isolated from the same pediatrics unit within a span of 2 months in the preceding year. Most of these (8 of 10  E. gallinarum , and all 3  E. casseliflavus ) isolates were from cases of septicemia. Although molecular epidemiological studies have not been done to compare the genetic similarities of these isolates, the data depicts the nosocomial spread of these species. WCP analysis by SDS-PAGE had been proven to assist in validating the species identities as well, to identify strains that do not exhibit phenotypic characteristics identical to the type strains of each species [ 4 , 10 , 21 ]. We were able to validate the authenticity of the unusual species, and the exact taxonomic status of the atypical phenotypic variant strains identified by conventional biochemical testing as shown in Figure- 1 , using WCP fingerprinting by SDS-PAGE. Ciprofloxacin resistance was 63% among isolates (excepting  E. casselifalvus ) which proves that it may be successful only in treating enterococcal urinary tract infections [ 1 , 9 ], since most of our isolates were from bloodstream and other related specimens. None of the isolates were β-lactamase producer, but penicillin and ampicillin resistance were exhibited by 54.3% and 45.7% isolates. We suggest penicillin binding protein modification based resistance for our isolates as a basis for β-lactam resistance, as depicted previously [ 22 , 23 ], but markedly differs from other Indian studies [ 15 , 24 ] showing up to 50% β-lactamase associated resistance. The prevalence of high-level gentamicin resistance (43.4%) and high-level streptomycin (37%) among unusual enterococcal isolates from our study partially correlates with studies from Japan [ 25 ] and United States [ 12 , 26 ]. In our study, most strains with high-level gentamicin resistance lacked high-level streptomycin resistance, and vice versa, thus facilitating the combination therapy (cell wall inhibitor plus aminoglycoside) treatment options for serious enterococcal bloodstream infections [ 1 , 9 ]. The prevalence of vancomycin resistance was 24% by agar screening /agar dilution method and 13% by disk diffusion. The difference may be attributed to the intrinsic low level vancomycin resistance (van C genotype), exhibited by 4  E. gallinarum  and, 1  E. casseliflavus  isolates, which may go undetected by disk diffusion testing [ 27 ]. Of serious concern was the low-level vancomycin resistance exhibited by one  E. durans  and two  E. mundtii  (MIC ≤ 6 μg/ml). The genotypic basis of vancomycin resistance for these 3 isolates yet to be studied, will give us a definitive picture regarding its clinical significance, since studies have reported the prevalence of vancomycin resistance in these two species, and its transferable nature from  E. durans  to  E. faecium  [ 28 - 30 ].
Discussion
Our study reveals that the prevalence of unusual species of enterococci as 19% in our clinical setup in South India. Many studies and reviews show the prevalence of non-faecalis and non-faecium enterococci as 2–10% [ 3 , 6 , 12 ]. Previous studies from India have reported  E. faecalis  and  E. faecium  as the only prevalent species [ 13 - 16 ], which may not reflect the true incidence rate. From our perspective the real incidence tends to be higher which in part can be explained as, misidentification of species due to exhibition of aberrant sugar reactions by some enterococci or, due to lack of application of the complete range of tests to identify non-faecalis and non-faecium enterococci [ 7 , 17 ]. The prevalence rate (19%) of our study was partly in accordance with another Indian study [ 18 ] showing 14.8% (excluding  E. faecalis  and  E. faecium ) prevalence of unusual species of enterococci from catheterized patients with urinary tract infections.  E. mundtii  and  E. durans  were not reported in their study, whose prevalence was 1.7% and 0.8% respectively in our study.  E. gallinarum  (6.2%) and  E. avium  (4.1%) were the most commonly identified species, which markedly differs in isolation rate (0.3–1.2%) from other studies [ 6 , 19 , 20 ]. The incidence of infections caused by unusual enterococcal species is of serious concern, since 43.5% of the isolates were from cases of septicemia without endocarditis. Apart from septicemia, the unusual species of enterococci were isolated frequently from cases of urinary tract infections, surgical and non-surgical wound infections and peritonitis. Most of the patients with the bloodstream infections had a peripheral or central catheter. Further, only 13% of enterococcal infections were polymicrobial, with majority from non-bloodstream isolates that underscores the clinical significance of these unusual enterococcal species. Although the unusual species of enterococci were isolated at regular intervals throughout our study period, we could find clustering of specific species during a specific time period from specific units/wards. Interestingly, 10 among the 15 isolates of  E. gallinarum  isolated during our study period were from pediatrics unit, while 7 of the 10 isolates exhibiting a similar antibiotype were isolated from the same ward within a span of 2 months. The remaining 3 of the 10  E. gallinarum  were isolated from the same ward in the preceding 3 months, one of which showed an antibiotype similar to the cluster of 7 isolates. The same was the case of 3  E. casseliflavus  isolated from the same pediatrics unit within a span of 2 months in the preceding year. Most of these (8 of 10  E. gallinarum , and all 3  E. casseliflavus ) isolates were from cases of septicemia. Although molecular epidemiological studies have not been done to compare the genetic similarities of these isolates, the data depicts the nosocomial spread of these species. WCP analysis by SDS-PAGE had been proven to assist in validating the species identities as well, to identify strains that do not exhibit phenotypic characteristics identical to the type strains of each species [ 4 , 10 , 21 ]. We were able to validate the authenticity of the unusual species, and the exact taxonomic status of the atypical phenotypic variant strains identified by conventional biochemical testing as shown in Figure- 1 , using WCP fingerprinting by SDS-PAGE.E. faecalisE. faeciumE. faecalisE. faeciumE. mundtiiE. duransE. gallinarumE. aviumE. gallinarumE. gallinarumE. casseliflavusE. gallinarumE. casseliflavusCiprofloxacin resistance was 63% among isolates (excepting  E. casselifalvus ) which proves that it may be successful only in treating enterococcal urinary tract infections [ 1 , 9 ], since most of our isolates were from bloodstream and other related specimens. None of the isolates were β-lactamase producer, but penicillin and ampicillin resistance were exhibited by 54.3% and 45.7% isolates. We suggest penicillin binding protein modification based resistance for our isolates as a basis for β-lactam resistance, as depicted previously [ 22 , 23 ], but markedly differs from other Indian studies [ 15 , 24 ] showing up to 50% β-lactamase associated resistance. The prevalence of high-level gentamicin resistance (43.4%) and high-level streptomycin (37%) among unusual enterococcal isolates from our study partially correlates with studies from Japan [ 25 ] and United States [ 12 , 26 ]. In our study, most strains with high-level gentamicin resistance lacked high-level streptomycin resistance, and vice versa, thus facilitating the combination therapy (cell wall inhibitor plus aminoglycoside) treatment options for serious enterococcal bloodstream infections [ 1 , 9 ]. The prevalence of vancomycin resistance was 24% by agar screening /agar dilution method and 13% by disk diffusion. The difference may be attributed to the intrinsic low level vancomycin resistance (van C genotype), exhibited by 4  E. gallinarum  and, 1  E. casseliflavus  isolates, which may go undetected by disk diffusion testing [ 27 ]. Of serious concern was the low-level vancomycin resistance exhibited by one  E. durans  and two  E. mundtii  (MIC ≤ 6 μg/ml). The genotypic basis of vancomycin resistance for these 3 isolates yet to be studied, will give us a definitive picture regarding its clinical significance, since studies have reported the prevalence of vancomycin resistance in these two species, and its transferable nature from  E. durans  to  E. faecium  [ 28 - 30 ].E. casselifalvusE. gallinarumE. casseliflavusE. duransE. mundtiiE. duransE. faeciumConclusion Precise identification of enterococci to species level enables us, to access the species-specific antimicrobial resistance characteristics, apart from knowing the epidemiological pattern and their clinical significance in human infections. The difficulty in detecting (intrinsic) low-level vancomycin resistance by disk diffusion testing [ 28 ] emphasizes the necessity for including agar screening methods as per NCCLS guidelines in routine susceptibility testing of all enterococci isolated from clinical specimens [ 9 ]. Further as shown in our study, the increase in the rate of prevalence of the unusual and atypical species and the emergence of multidrug resistance among them, highlights the significance of rapid and accurate identification of enterococci to the species level for initiating appropriate therapeutic regimen, and reemphasizes the importance of the implementation of appropriate infection control measures to limit the nosocomial spread of these unusual species in any nosocomial setting.
Conclusion
Precise identification of enterococci to species level enables us, to access the species-specific antimicrobial resistance characteristics, apart from knowing the epidemiological pattern and their clinical significance in human infections. The difficulty in detecting (intrinsic) low-level vancomycin resistance by disk diffusion testing [ 28 ] emphasizes the necessity for including agar screening methods as per NCCLS guidelines in routine susceptibility testing of all enterococci isolated from clinical specimens [ 9 ]. Further as shown in our study, the increase in the rate of prevalence of the unusual and atypical species and the emergence of multidrug resistance among them, highlights the significance of rapid and accurate identification of enterococci to the species level for initiating appropriate therapeutic regimen, and reemphasizes the importance of the implementation of appropriate infection control measures to limit the nosocomial spread of these unusual species in any nosocomial setting.Competing interests The author(s) declare that they have no competing interests.
Competing interests
The author(s) declare that they have no competing interests.Authors' contributions PVP designed the study and carried out the experimental works and analysis, and drafted the manuscript. RSR supervised and participated in the design of the study and coordination, and helped to draft the manuscript. SCP participated in the coordination of the study and helped to draft the manuscript.
Authors' contributions
PVP designed the study and carried out the experimental works and analysis, and drafted the manuscript. RSR supervised and participated in the design of the study and coordination, and helped to draft the manuscript. SCP participated in the coordination of the study and helped to draft the manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here: