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Pathogen profile and drug resistance analysis of spontaneous peritonitis in cirrhotic patients.

AIM
To investigate the microbiological characteristics and drug resistance in liver cirrhosis patients with spontaneous peritonitis.


METHODS
We analyzed the data of patients with liver cirrhosis and abdominal infection at the First Affiliated Hospital of Zhejiang University between January 2011 and December 2013. Pathogens present in the ascites were identified, and their sensitivity to various antibiotics was determined.


RESULTS
We isolated 306 pathogenic bacteria from 288 cases: In 178 cases, the infection was caused by gram-negative strains (58.2%); in 85 cases, gram-positive strains (27.8%); in 9 cases, fungi (2.9%); and in 16 cases, more than one pathogen. The main pathogens were Escherichia coli (E. coli) (24.2%), Klebsiella pneumoniae (18.9%), Enterococcus spp. (11.1%), and Staphylococcus aureus (7.5%). Of the 306 isolated pathogens, 99 caused nosocomial infections and 207 caused community-acquired and other infections. The E. coli and K. pneumoniae strains produced more extended-spectrum β-lactamases in cases of nosocomial infections than non-nosocomial infections (62.5% vs 38%, P < 0.013; 36.8% vs 12.8%, P < 0.034, respectively). The sensitivity to individual antibiotics differed between nosocomial and non-nosocomial infections: Piperacillin/tazobactam was significantly more effective against non-nosocomial E. coli infections (4% vs 20.8%, P < 0.021). Nitrofurantoin had stronger antibacterial activity against Enterococcus species causing non-nosocomial infections (36.4% vs 86.3%, P < 0.009).


CONCLUSION
The majority of pathogens that cause abdominal infection in patients with liver cirrhosis are gram-negative, and drug resistance is significantly higher in nosocomial infections than in non-nosocomial infections.


INTRODUCTION
Decompensated liver cirrhosis affects 25% to 47% of the population, and ascites is the most common complication; it is characterized by decreased immune function, which consequently leads to abdominal cavity, blood, lung, urinary tract and other infections that worsen with time and have a high fatality rate [1,2] . Studies have shown that decreased immunity combined with weak intestinal motility, increased intestinal permeability, reduced secretion of intestinal antimicrobial peptides and other factors are likely to lead to bacterial translocation in patients with decompensated cirrhosis [3][4][5] . Spontaneous bacterial peritonitis is a common infection; it causes further deterioration of liver function, multi-organ failure and sepsis, which are associated with poor prognosis and a low survival rate [6][7][8][9] . The mortality rate associated with multiple organ failure and septicopyemia caused by spontaneous bacterial peritonitis is still over 75% [10][11][12] . Therefore, antibiotic therapy for spontaneous peritonitis has become an important means of improving the survival rate in patients with cirrhosis.
Third-generation cephalosporins are mainly used for the treatment of spontaneous bacterial peritonitis; however, it was shown that cefotaxime, one of the most commonly used cephalosporins, had a lower success rate than expected [13] . This is probably attributable to the emergence of antibiotic-resistant pathogen strains. While earlier reports showed that 70% of the cases of spontaneous bacterial peritonitis were caused by gram-negative bacteria such Escherichia coli (E. coli) and Klebsiella pneumoniae [14] , this bacterial profile has begun to change and the emergence of resistant bacterial species has been indicated. In recent years, the incidence of grampositive abdominal infections in patients with decompensated cirrhosis has increased significantly; this phenomenon is attributed to the use of antibiotics that alter the intestinal flora such that it is conducive to the translocation of gram-positive bacteria [15][16][17] . Another study has attributed the increasing incidence of gram-positive bacterial peritonitis to increased resistance to quinolones, such as norfloxacin, which are also commonly used for the treatment of this infection [18] . Thus, it is important to study in detail the pathogen profiles in spontaneous bacterial peritonitis and resistance of the pathogens to currently used antibiotics. Based on this, clinicians can select the appropriate antibiotic treatment to control the emergence and development of pathogenic bacteriaresistant strains in intra-abdominal infections.
Although some studies have examined the pathogen profiles and drug resistance in spontaneous peritonitis [19,20] , there is definitely a need for new and comprehensive data on the same. Therefore, in this study, we retrospectively surveyed and analyzed the types of pathogens and their drug resistance in the ascites culture samples of 288 patients who had cirrhosis with abdominal infection at our hospital between January 2011 and December 2013.

Clinical data
The medical ethics committee at The First Affiliated Hospital of Zhejiang University School of Medicine approved the protocol for the retrospective study before the clinical data were collected. All cirrhotic patients at our hospital between January 2011 and December 2013 were retrospectively analyzed. Each case history was registered using a unified registration form: the information included gender; age; duration of hospitalization; medical history; clinical manifestations; hospitalization course; disease prognosis; and the results of abdominal ultrasonography, abdominal paracentesis, routine ascites test, ascites culture, liver function test, kidney function test, blood coagulation test, and routine blood examination. If the ascites culture results obtained following abdominocentesis at 48 h after admission was positive, this was considered to indicate a nosocomial infection. If a patient repeatedly tested positive for the same pathogen in the ascites culture examination, only the first result was recorded. The criteria for the diagnosis of spontaneous bacterial peritonitis were positive ascites culture for bacteria and neutrophil count in the ascites sample greater than 250/mm 3 [21] .

Susceptibility testing
The Kirby-Bauer (KB) method was used for drug sensitivity testing on Müller-Hinton agar.

Statistical analysis
The variables were counting variables, and the description of the count data is described by a ratio or the rate of the data. The t test was used to analyze measurement data, and the χ 2 test was used to analyze count data. P < 0.05 was considered to indicate statistical significance. SPSS 17.0 was used for all the statistical analyses.

Clinical characteristics
The clinical data of 6086 patients with liver cirrhosis were gathered over the three-year study period. Of these patients, 506 had abdominal infections, excluding secondary abdominal infections (ascites fluid neutrophil count was ≥ 250/mm 3 , a positive ascites fluid culture, and evidence of an intra-abdominal source of infection, demonstrated by surgery, autopsy, or abdominal CT) [22] , according to the results of the ascites neutrophil count and bacterial culture. Spontaneous bacterial peritonitis was identified in 288 cases (198 male and 90 female patients; age range, 23-79 years; mean age, 55 ± 12.6 years), and 306 pathogens were isolated. Most patients had advanced liver cirrhosis (68.9%, Child C; 28.6%, Child B; and 2.5%, Child A); the mean Child score was 10.5. A few patients (less than 10%) had received preventive quinolone treatment. Among the 288 patients, 208 had abdominal pain and fever, 22 had septic shock and 42 had multiple organ dysfunction syndrome. Fiftyone of the patients died at the end of the treatment.
In 137 cases, the WBC count was ≥ 10.0 × 10 9 and in 45 cases, the WBC count was ≤ 4.0 × 10 9 . . ESBL was produced in more cases of nosocomial infections than in non-nosocomial infections, and the sensitivity of individual antibiotics differed between nosocomial and non-nosocomial infections: Ampicillin/sulbactam and ceftriaxone were significantly more effective

DISCUSSION
In this study, we isolated 306 pathogens from ascites culture samples of 288 hospitalized patients with liver cirrhosis and abdominal infection over a period of 3 years: gram-negative bacteria formed the majority of the pathogens (58.2%), and mainly included E. coli, K. pneumoniae, A. baumannii, A. hydrophila, P. aeruginosa, Burkholderia cepacia and other glucose non-fermenting bacteria. Other studies have also shown that gram-negative bacteria formed the majority of pathogens in patients with liver cirrhosis complicated with spontaneous bacterial peritonitis [10,23] . However, the findings from these studies, including the present study, are in contrast to the more recent trends, which have shown an increase in the number of infections caused by gram-positive bacteria. For example, Alexopoulou et al [24] showed that 55% of the spontaneous bacterial peritonitis cases in cirrhotic patients were caused by gram-positive cocci. Furthermore, Piroth et al [25] showed that gram-positive cocci formed the majority of pathogenic bacteria in cases of spontaneous bacterial peritonitis and ascites; the authors supposed that this was related to the use of prophylactic antibiotics in patients with liver cirrhosis (which altered the intestinal flora), the use of invasive procedures and other factors. Our data showed that the use of prophylactic antibiotics was not too high in our group of patients with liver cirrhosis, which probably explains why more gram-negative than gram-positive bacteria were isolated. Of the 306 isolated pathogens, 99 were responsible for hospital-acquired infections (32.4%); this finding was similar to that of Ariza et al [26] , who found that 40.9% of cases of spontaneous bacterial peritonitis were nosocomial infections. Our patient population comprised a high number of cases with advanced Table 3

Comparison of the drug resistance of Escherichia coli to commonly used antibacterial agents between nosocomial and nonnosocomial spontaneous bacterial peritonitis n (%) Antibacterial agents Non-nosocomial infections (n = 50) Nosocomial infections (n = 24) P value (χ 2 test)
ESBL test 19   cirrhosis (70.9% with Child C type). They had severe fever, abdominal pain and other symptoms of peritonitis. In our study, the most predominant pathogen was E. coli (24.2%), followed by K. pneumoniae (18.9%) and then enterococci (11.1%). Moreover, the percentages of MRSA and MRCNS were 26.1% and 29.4% respectively. These findings are similar to some recent and older studies on the pathogen profile of spontaneous bacterial peritonitis [19,20,24,25] . Furthermore, fungal pathogens caused 2.9% of the infections, which was similar to a previous report [27] . It is therefore   important to start anti-fungal treatment as soon as fungal species are discovered in the ascites culture of cirrhotic patients with peritonitis. Our data showed that resistance of the major gramnegative as well as gram-positive bacteria to thirdgeneration cephalosporins was higher in nosocomial infections than in non-nosocomial infections, which is in agreement with Song et al [28] 's findings and a recent study by Sheikhbahaei et al [20] . Imipenem, meropenem, piperacillin/tazobactam, and amikacin had good antibacterial activity against E. coli and K. pneumoniae in abdominal infections (sensitivity rate, ≥ 86.2%). However, E. coli and K. pneumoniae showed high resistance against gentamicin, ampicillin and cephalosporins; this is in keeping with the recent trend observed by Park et al [19] . Therefore, the findings indicate that preference should be given to the first group of antibiotics when treating cirrhotic patients with peritonitis caused by E. coli and K. pneumoniae strains. Furthermore, piperacillin/tazobactam showed significantly stronger activity against non-nosocomial infections caused by E. coli, and gentamicin, cefoperazone/sulbactam, ceftriaxone, and ampicillin/ sulbactam showed stronger activity against nonnosocomial infections caused by K. pneumoniae.
The sensitivity of P. aeruginosa and Acinetobacter spp. to the antibiotic agents was low, but Acinetobacter spp. had stronger drug tolerance than P. aeruginosa. The drug resistance of gram-negative bacteria against quinolone antibiotics was higher than that of grampositive bacteria, particularly in cases of nosocomial infections, which is consistent with the results of previous studies [29] . The detection rate of ESBL-producing E. coli was higher than that of ESBL-producing K. pneumoniae (45.9% vs 20.7%). Moreover, ESBL production was higher in cases of nosocomial infections than in cases of non-nosocomial infections. Previous studies have also shown that ESBL production was higher in cirrhotic patients with nosocomial infection than in those with non-nosocomial infection; ESBL production improves bacterial resistance and consequently results in increased mortality [10,30] . Our results showed a significant difference in the meropenem resistance of E. coli between nosocomial and nonnosocomial infections; this was probably related to the widespread use of carbapenems. The aminoglycoside and β-lactamase inhibitor complex had higher activity against ESBL-producing E. coli and K. pneumoniae strains. Although glucose non-fermenting bacteria have higher sensitivity to commonly used antibiotics, the detection rate of non-fermenting bacteria in clinical specimens has been increasing every year. This trend is probably related to the use of mechanical ventilator-assisted ventilation, atomization devices, cardiopulmonary bypass, various types of catheters, and the long-term use of high doses of cephalosporins, carbapenems and other antibacterial drugs; thus, non-fermenting bacteria are important pathogens in nosocomial infection [31,32] . Therefore, patients with cirrhosis who are likely to have abdominal infection should be treated with the enzyme complex comprising aminoglycoside and β-lactamase inhibitors and diene hydrocarbon enzyme antibacterials that have strong activity against gram-negative bacteria. Aminoglycoside antibiotics have high renal toxicity and weak activity against a number of drug-resistant strains, so they are not usually preferred for the treatment of cirrhosis. The antimicrobial activity of vancomycin, nitrofurantoin and rifampin against Staphylococcus spp. in abdominal infections was strong (sensitivity rate, ≥ 87%). Furthermore, the susceptibility of Staphylococcus spp. to gentamicin, clindamycin and moxifloxacin was also strong (sensitivity rate, ≥ 60.9%). Enterococcus was resistant to a variety of antibacterial agents, but showed high sensitivity to vancomycin, rifampin, clindamycin and gentamicin (sensitivity rate, ≥ 70.6%). Furthermore, nitrofurantoin showed stronger activity against Enterococcus spp. in non-nosocomial infections than in nosocomial infections. Vancomycin, rifampin, moxifloxacin and nitrofurantoin had strong antibacterial activity against S. pneumoniae (sensitivity rate, ≥ 71.9%); ofloxacin, levofloxacin, moxifloxacin and clindamycin had greater activity against S. aureus strains in non-nosocomial infections. Thus, when there is evidence of Staphylococcus spp. in abdominal infections, glycopeptide antibiotics should be the preferred medication.
Cirrhosis with secondary bacterial infection in patients is an important cause of liver function deterioration and other complications, thus early diagnosis and prompt treatment are important [2,33] . This study is a retrospective analysis, so there are some selection biases and limitations due to the limited sample size. Nonetheless, the findings are important and they lay the foundation for future improvements in the culture, sensitivity tests and the antibiotic regime for spontaneous bacterial peritonitis in cirrhotic patients.

Background
Spontaneous bacterial peritonitis (SBP) is a common and severe complication in cirrhotic patients with ascites. Monitoring drug resistance in cases of abdominal infection with liver cirrhosis is important for establishing the appropriate antibiotic regimes, reducing the generation of drug-resistant bacteria and reducing the associated mortality.

Research frontiers
Changes in the epidemiology of the causative bacteria in SBP and bacterascites and in their susceptibility to antibiotics have been associated with low treatment efficacy of traditional therapy in nosocomial infections.

Innovations and breakthroughs
This study addressed two primary issues regarding spontaneous peritonitis in cirrhotic patients in China: first, the pathogen profile and, second, drug resistance. The majority of pathogenic bacteria that cause abdominal infection in liver cirrhosis are gram-negative, and drug resistance is significantly higher in