Bacterial Infection in Liver Cirrhosis: the Microbiologist Point of View Prof. Marie-Hélène NICOLAS-CHANOINE

Bacterial infections life-threatening complications in cirrhotic patients and common

30 to 50 % of hospitalized cirrhotic patients are concerned by bacterial infections

Spontaneous Bacterial Peritonitis (SBP) (± bacteremia) Urinary Tract Infection (UTI) (± bacteremia) Pulmonary infection Others (peritoneal tuberculosis ) 25 % of death directly due to bacterial infection

Host risk factors for SBP Surviving to a previous SBP episode Low ascitic fluid protein levels (<10g/L) Gastrointestinal hemorrhage

Physiopathology of SBP SBP is caused by intestinal micro-organisms that translocate through the mucosal barrier to the mesenteric lymph nodes, enter the bloodstream and reach the ascitic fluid.

ParameterNumber (%) CommunityNosocomialTotal Episode number Isolate number Plurimicrobial25 7 (3.5) Enterobacteriacae E.coli 61 (47)27 (36)88 (43) Klebsiella spp268 Others41014 Streptococci 52 (40)15 (20)67 (32) Viridans group S. bovis9211 Pneumococci729 B group808 S. aureus257 Enterococcus spp15 6 (3) Others 7310 Candida23 5 (2.5) Bacterial species isolated from AF obtained from patients with SBP and hospitalized in Beaujon hospital ( )

Are bacterial factors involved in morbidity or/and mortality in cirrhotic patients with SBP? Genetic background of Escherichia coli isolates from patients with spontaneous bacterial peritonitis: relationship with host factors and prognosis. F. Bert et al, Clin. Microbiol. Infect. (in press)

- 4 phylogenetic groups: A, B1, B2 and D - extraintestinal pathogens: more often group B2 isolates - virulence factors (VF)-encoding genes - group B2 isolates have more VF genes than non B2 group isolates Population structure of E. coli

VF genePrevalence of VF gene, No. (%) of isolates Group A (n=20) Group B1 (n=3) Group B2 (n=35) Group D (n=18) Total (n=76) Adhesins papC7 (35)026 (74)6 (33)39 (51) papG allele II0015 (43)6 (33)21 (28) papG allele III0011 (31)011 (14) sfa/foc0018 (51)018 (24) Toxins hly0018 (51)1 (5.6)19 (25) cnf10017 (49)0 17 (22) Siderophores fyuA8 (40)1 (33)35 (100) 11 (61)55 (72) aer 10 (50)2 (66)19 (54) 12 (67)43 (56) Prevalence of virulence factor (VF) genes according to phylogenetic groups in 76 E. coli isolates from patients with SBP ( ) Mean VF score of B2 versus non B2: 15.4 vs 7.3 p<10 -4

Value in the indicated group* VariablePatients with B2 isolates (n= 35) Patients with non-B2 isolates (n= 41)P Age (year)55 NS Male gender26 (75)34 (83)NS Alcoholism22 (65)25 (64)NS Viral hepatitis8 (24)11 (28)NS Hepatocellular carcinoma7 (20)4 (10)NS Previous SBP episode2 (6)12 (30) Norfloxacin prophylaxis1 (3)9 (22) MELD score Blood neutrophils (cells/mm3)10,7527,931NS Platelet (cells/m 3 )136,828100, Prothrombin ratio (%) Serum bilirubin (μmol/L)200178NS Serum creatinine (μmol/L)142182NS Serum sodium (μmol/L)131132NS AF neutrophils (cells/mm 3 )4,3894,501NS AF protein (g/L) Hospital-acquired SBP11 (31)16 (39)NS Positive blood cultures9 (26)15 (37) Comparison of host factors in patients with B2 isolates and those with non-B2 isolates. * data are no (%) of patients or mean value ; NS, non significant (p 0.2) ; SBP, spontaneous bacterial peritonitis AF, ascitic fluid, red indicates host factors independently associated with non-B2 isolates

10/76 (13%) patients with fluoroquinolone prophylaxis Prevalence of fluoroquinolone resistance in the 76 SBP E. coli = 16% Fluorouinolone resistance significantly higher in patients with norfloxacin prophylaxis than in those without :70% vs 7.6%, p <10 -4 Fluoroquinolone resistance significantly higher in non B2 isolates than in B2 isolates: 30% vs 0%, p <0.001

Overall, we found that the prevalence of non B2 isolates (fewer VF and more often resistant) increased with the severity of liver disease

VariableORCI 95 %P MELD score – Hospital-acquired SBP – Prothrombin ratio – Serum creatinine level – Hospital-acquired SBP – Multiple logistic regression of risk factors for in-hospital mortality 1 1: the first multivariate analysis tested the MELD score and the second multivariate analysis tested the components of the score, 2: value for an increase of 5, 3: value for a decrease of 10 %, 4: value for an increase of 50 μmol/L

Host factors, namely the severity of renal and hepatic dysfunctions outweigh bacterial factors in predicting SBP in-hospital mortality

Viridans Streptococci

Viridans group streptococci (VGS) in 56 episodes*of SBP and/or bacteremia in 51 patients** ( ) Species SBP (n = 39)*** Bacteremia without SBP (n = 17) S. oralis146 S. mitis101 S. salivarius46 S. gordonii33 S. sanguis30 S. vestibularis30 S. mutans01 others20 * 60,7 % acquired in the community,** 5 patients with 2 consecutive episodes *** 4 episodes with bacteremia Liver Transplantation (in press)

Antibiotic susceptibility of the 56 VGS Ten patients had a prior episode of SBP and were receiving norflaxacin prophylaxis. No VGS resistant to fluoroquinolones. penicillin: 71 % amoxicillin: 87.5 % cefotaxime: 89.3 % erythromycin: 59 % levofloxacin: 100 % moxifloxacin: 100 %

Demographic and biological data in 115 episodes of SBP caused by viridans group streptococci or E. coli Variable SBP caused by p VGS (n = 39)E. coli (n = 76) Age (year) NS Male gender30 (76.9 %)60 (78.9 %)NS Alcoholism18 (46.2 %)46 (63 %)NS Viral hepatitis17 (43.6 %)19 (36 %)NS Carcinoma7 (17.8 %)11 (14.5 %)NS MELD score <0.01 Norfloxacin prophylaxis9 (23.1 %)10 (13.2 %)NS Blood PMN (cells/mm 3 )7,6728,850NS AF PMN (cells/mm 3 )1,4264,451<0.001 AF protein (g/L) NS Nosocomial origin13 (33.3 %)24 (31.6 %)NS Positive blood cultures4 (10.5 %)29 (35.5 %)< day mortality9 (23.1 %)27 (38 %)*NS NS, non significant; PMN, polymorphonuclear leucocytes; AF, ascitic fluid.* Data available for 71 patients.

Multi drug-resistance in E. coli related to extended-spectrum ß-lactamase (ESBL) production, notably CTX-M enzymes

Endémic CTX-M-1CTX-M-8CTX-M-9 Sporadic CTX-M CTX-M-2, - 5 CTX-M-16, -17 CTX-M-8 CTX-M-9, -16 CTX-M-1, 3, 15 CTX-M-9, -14, 18, 19, 20, 21 CTX-M-2, -5 CTX-M-, 3, 15CTX-M-2TOHO-like CTX-M-2 CTX-M-3, 15 CTX-M-14 CTX-M-3, 15 CTX-M-9, -13, -14 CTX-M-3 CTX-M-9,-14 CTX-M-1,10,15 CTX-M-4, -6 CTX-M-3 CTX-M-15 CTX-M-9,-14 CTX-M-1,10,15,32 Lewis J, AAC 2007, « CTX-M-type as the predominant ESBL isolated in a US health care system » (dominance of CTX-M-15) Canton R. Curr. Opin. Microbial. 2006

Groupe B2 Resistance to fluoroquinolones Lower number of VF-encoding genes than expected in B2 isolates

Canada France Spain England Turkey India Portugal Switzerland Korea

ESBL-producing E.coli and cirrhotic patients ? Still rare as agent responsible for SBP / bacteremia - 2 patients, June and Sept 2007 at Beaujon hospital - Korean J Hepatol sept 2007: survey on 12 years, emergence of ESBL-producing E. coli but carried in the digestive tract (rectal swabs)

Period Incidence / 100 screened patients Hepatology*ICU**Hospital 7/2 – 6/ /6 – 15/1242 Beaujon Hospital (2006): incidence of fecal ESBL-positive enterobacteriaceae * patients screened at admission,** patients screened at admission, then once a week 8 patients with ESBL-producing E. coli, 5 CTX-M-15 and 2 isolates belonging to clone ST131

In 2008 Good and bad news about clinical and microbiological data with regard to SBP Good news: norfloxacin prophylaxis not only decreases the risk of second SBP but also delays hepato-renal syndrome and improves survival in cirrhosis. Fernandez J et al, Gastroenterology Sep;133(3): Bad news. E. coli is become the enterobacterial species the most concerned by ESBL and fluoroquinolone resistance is extremely frequent in those E. coli producing CTX-M enzyme

Frederic Bert: infection in cirrhotic patients and patients with liver transplant Véronique Leflon Guibout: molecular mechanisms of resistance and molecular epidemiology Latifa Noussair: Mycobacterium tuberculosis infection diagnosis including tuberculosis peritonitis in cirrhotic patients

CharacteristicValue* Epidemiological features Age (yr) 54.7 ± 10.6 Male gender 60 (78.9) Alcoholism 46 (63) Viral Hepatitis 19 (36) Carcinoma 11 (14.5) Previous SBP episode 14 (18.4) Norfloxacin prophylaxis 10 (13.2) Meld score 27.9 ± 9.7 Blood variables PMN (cells/mm 3 ) 8,850 ± 5,989 Platelet (cells/mm 3 ) 117,000 ± 85,618 Prothrombin ratio (%) 35.9 ± 15.6 Bilirubin (µmol/L) 188 ± 138 Creatinine (µmol/L) 163 ± 140 Sodium (mmol/L) 131 ± 5.6 Ascitic fluid variables PMN (cells/mm 3 ) 4,451 ± 4,720 Total protein (g/L) 10.4 ± 5.1 Characteristics of cirrhotic patients in 76 episodes of spontaneous bacterial peritonitis (SBP) * Data are means ± SD or numbers (%) of patients

Prevalence of group or VF gene, no. (%) Trait Ciprofloxacin-susceptible (n= 64) Ciprofloxacin-resistant (n=12) Phylogenetic group A13 (20.3) 7 (58.3) B12 (3.1) 1 (8.3) B235 (54.7) 0 D14 (21.9) 4 (33.3) VF genes papC34 (53.1) 5 (41.7) papGII21 (32.8) 0 papGIII11 (17.2) 0 sfa/foc18 (28.1) 0 hly19 ( 29.7) 0 cnf117 (26.6) 0 fyuA49 (76.6) 6 (50) aer36 (56.2) 7 (58.3) Distribution of phylogenetic groups and virulence factor (VF) genes in relation to susceptibility to ciprofloxacin

Value in the indicated group* VariablePatients who died (n= 38)Patients who survived (n= 33)P Age (year)51.753NS Male gender31 (81.6)25 (75.7)NS Alcoholism21 (58.3)22 (68.7)NS Viral hepatitis10 (27.8)8 (25)NS Hepatocellular carcinoma6 (15.8)5 (15.1)NS Previous SBP episode5 (13.2)8 (24.2)NS Norfloxacin prophylaxis3 (7.9)6 (18.2)NS MELD score Blood neutrophils (cells/mm 3 )10,2937, Platelet (cells/m 3 )117,552114,180NS Prothrombin ratio (%) Serum bilirubin (μmol/L) Serum creatinine (μmol/L) Serum sodium (μmol/L) NS AF neutrophils (cells/mm 3 )4,9924,181NS AF protein (g/L) NS B2 group16 (48.1)17 (51.5)NS VF score2.9 NS Amoxicillin resistance21 (55.3)14 (42.4)NS Amoxi-clavulanate resistance5 (13.2)5 (15.1)NS Cefatoxime resistance1 (2.6)1 (3)NS Ciprofloxacin resistance5 (13.2)6 (18.2)NS Cotrimoxazole resistance14 (36.8)7 (21.2) Hospital-acquired SBP16 (42.1)7 (21.2) Positive blood cultures16 (42.1)8 (24.2) Appropriate empiric antibiotics33 (94.3)30 (96.7)NS Albumin therapy9 (23.7)11 (33.3)NS Unvariate analysis of host and bacterial factors associated with in-hospital mortality * data are no (%) of patients or mean value ; NS, non significant (p 0.2) ; SBP, spontaneous bacterial peritonitis ; AF, ascite fluid ; VF, virulence factor

Bacteremia without SBP (n = 17)* AsciteNumber without3 Sterile ascite9 Bacterascites (PNM < 250 mm 3 )5 * one patient with endocardites primary bacteremia = 16