Introduction Extended spectrum beta-lactamase (ESBL)-producing bacilli. Methods. KPP Abhilash 1, Balaji Veeraraghavan 2, OC Abraham 1.

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1 SUPPLEMENT TO JAPI december 2010 VOL Epidemiology and Outcome of Bacteremia Caused by Extended Spectrum Beta-Lactamase (Esbl)-producing Escherichia Coli and Klebsiella Spp. in a Tertiary Care Teaching Hospital in South India KPP Abhilash 1, Balaji Veeraraghavan 2, OC Abraham 1 Abstract Background: Production of extended spectrum beta-lactamase (ESBL) is one of the most important resistance mechanisms that hamper the antimicrobial treatment of infections caused by Enterobacteriaceae. Therefore, it is imperative to quantify the problem, and reinforce guidelines promoting appropriate antibiotic use. Objectives: To determine the prevalence, risk factors and the outcome of antibiotic treatment among hospitalized adults with bacteremia caused by ESBL producing strains of E. coli and Klebsiella spp. Study design: Prospective cohort study Methods: Sequentially encountered patients bacteremias due to E. coli or Klebsiella spp. were prospectively followed up for 14 days from the diagnosis of bacteremia. Results: Among the 131 bacteremic patients (62.6% nosocomially acquired), ESBL production was detected in 73.28% of the isolates of E. coli and Klebsiella spp. ESBL production was more common among isolates from patients with nosocomial infections than isolates from community acquired infections (85.37% versus 53.06%; p= <0.001). Prior use of 3 rd or 4 th generation cephalosporins was associated with an increased risk of ESBL production (p=0.017). A high degree resistance to multiple classes of antibiotics was noted. Carbapenems were the most active antibiotics in-vitro (imipenem susceptibility 99.2%). The commonest source of bacteremia was the urinary tract (45.04 %). The 14-day mortality rate was 23.6%. There was no significant difference was seen in the mortality rate between E. coli and Klebsiella spp. infections, ESBL-producing and non-esbl-producing strains, nosocomial and community acquired infections and among those treated with inappropriate antibiotics initially. Conclusions: This study shows a very high ESBL production and resistance to multiple classes of antibiotics, even among patients with community acquired infections caused by Enterobacteriaceae. The empiric use of 3 rd and 4 th generation cephalosporins should be curtailed, as cephalosporin use was associated with an increased risk of ESBL production. In view of their excellent in-vitro activity, carbapenems should be the initial empiric choice for serious life threatening infections caused by ESBL producing Enterobacteriaceae, with prompt de-escalation when culture and susceptibility results become available. Introduction Extended spectrum beta-lactamase (ESBL)-producing bacilli from the family Enterobacteriaceae are increasingly identified as pathogens, having become endemic in many healthcare settings. ESBLs are plasmid mediated beta-lactamases capable of efficiently hydrolyzing many beta-lactam antibiotics including 3 rd generation cephalosporins and monobactams. Beta-lactamase inhibitors (clavulanic acid, sulbactam, and tazobactam) generally inhibit ESBL producing strains. The plasmids encoding ESBLs frequently encode for resistance to other class of antimicrobials also, thereby seriously limiting the choice of antimicrobials available for the treatment of these infections. The prevalence of ESBL producing strains among Gramnegative bacilli varies widely (1-3) depending upon the clinical setting and the geographic region. Risk factors for the occurrence of ESBL producing organisms include increased length of stay in the ward or intensive care unit, severity of illness, respiratory or urinary tract infections, malignancy, in-dwelling urinary catheter, dialysis, age > 65 years, functional dependence, emergency abdominal surgery, and prior administration of antimicrobials. 1 Departments of Medicine Unit 1 & Infectious Diseases, 2 Microbiology, Christian Medical College, Vellore Infections due to ESBL producing Enterobacteriaceae are often complicated due to the increased treatment cost, length of hospital stay, morbidity and mortality. We conducted this study to quantify the prevalence, risk factors and outcome among hospitalized patients with ESBL producing Enterobacteriaceae bacteremias, as this has not been systematically investigated in India. Methods This prospective cohort study was conducted at the Christian Medical College (CMC), Vellore, a 2200 bedded academic medical center in south India between 1 st February 2007 and 31 st May We recruited sequentially encountered patients older than 16 years with E. coli or Klebsiella spp. bacteremia who were admitted in various wards and intensive care units of CMC Hospital, Vellore. The inclusion criteria were isolation of E. coli or Klebsiella spp. from blood culture samples and willingness to participate in the study. Patients aged less than 16 years, outpatients and bacteremias of polymicrobial etiology were excluded. In all patients, only the first episode of bacteremia was included for further analysis. The patients were followed up for 2 weeks after the diagnosis of bacteremia to assess the clinical outcome.

2 14 SUPPLEMENT TO JAPI december 2010 VOL. 58 Table 1 : Baseline characteristics of patients with bacteremia (n = 131) Patient characteristics ESBL producing isolates (N = 96) Non-ESBL producing isolates (N = 35) Median age (years) Sex (M:F) 61:35 17: Primary source Urinary tract 44 (45.8%) 15 (42.9%) Intra abdominal 19(19.8%) 10 (28.6%) Pneumonia 7 (7.3%) Undetermined 26 (27.1%) 10 (28.6%) Nosocomial 70 (72.9%) 12 (34.2%) <0.001 infections Co-morbid conditions Type 2 diabetes 44 (45.8%) 12 (34.3%) Chronic renal 8 (8.3%) 3 (8.6%) failure Chronic liver 7 (7.3%) 6 (17.1%) disease Hypertension 21 (21.9%) 7 (20%) Solid tumors 7 (7.3%) 3 (8.6%) Hematological 14 (14.6%) 6 (17.1%) malignancy HIV infection 3 (3.1%) 1 (2.9%) Ischemic heart 5 (5.2%) 4 (11.4%) disease Cerebrovascular 2 (2.1%) 2 (5.7%) accident Recent surgery 17 (17.7%) 6 (17.1%) Prior antibiotic use Beta-lactam + Betalactamase inhibitors (n=7) Cephalosporins (3 rd and 4 th generation) (n=20) Fluoroquinolones (n=13) Aminoglycosides (n=15) Carbapenems (n=3) Any antibiotic (n=58) Initial antibiotic choice Beta-lactam + Betalactamase inhibitors Cephalosporins (3 rd and 4 th generation) Fluoroquinolones Aminoglycosides Carbapenems Others Initial antibiotic choice appropriate 42 (43.75%) 32 (91.42%) <0.001 A study questionnaire was formulated and duly filled with all relevant information related to risk factors that contribute to antimicrobial resistance, such as demographic details, severity of illness, co-morbidities, source of infection (community acquired or nosocomial), prior antibiotic use etc. During the study period the administration of antibiotics and other therapeutic agents were done by the treating physician and the choice was not p influenced by the study. Severity of acute illness at the time of positive blood culture was assessed by a previously validated scoring system, based on mental status, vital signs, need for mechanical ventilation, and recent cardiac arrest (Pitt bacteremia score). 4 Patients accumulating 4 or more points were defined as severely ill. The primary source of the bacteremia was defined as pneumonia, urinary tract infection, intra-abdominal infection, and primary bloodstream infection, according to Centers for Disease Control and Prevention definitions. 5 Microbiological methods: For all the patients enrolled in this study, blood (5-8 ml) was collected in adult blood culture bottle (BacT/Alert) using standard precautions and processed by semi-automated blood culture system (BacT/Alert; BioMérieux, Marcyľ Etoile, France). Identification of the causative organisms was performed by standard microbiological methods. 6 Antibiotic susceptibility testing (AST) was carried out using disk diffusion method. The interpretation was based on the recommendations of Clinical Laboratory Standards Institute (CLSI). E. coli American Type Culture Collection (ATCC) 25922, P. aeruginosa ATCC and S. aureus ATCC were used as quality controls. The screening for ESBL production was done as per the CLSI guidelines. Briefly, a zone of inhibition with 23 mm for ceftazidime (30 µg), 28 mm for cefotaxime (30 µg) and 28 mm for aztreonam (30 µg) against K. pneumoniae, K. oxytoca and E. coli was considered as suggestive of ESBL production. ESBL production (suggested by the screening test) was further phenotypically confirmed by double disk diffusion test as per CLSI protocol. For the confirmatory test, both cefotaxime and ceftazidime, alone and in combination with clavulanic acid were tested. The criteria for classifying as an ESBL-producing isolate was a 5 mm increase in a zone diameter for either antimicrobial agent tested in combination with clavulinic acid, opposed to its zone when tested alone. ATCC K. pneumoniae was used as quality control strain along with the study isolates. The quality criteria for ATCC K pneumoniae was 5 mm increase in ceftazidime-clavulanic acid zone diameter; 3 mm increase in cefotaxime-clavulanic acid zone diameter. 7 Statistical methods: Data entry was done using the Statistical Package for the Social Sciences (SPSS) software package (version 11). Descriptive statistics were calculated using SPSS software. Chi-square test was used for comparison of categorical variables. Odds ratio (OR) and confidence intervals (CI) were calculated and a p value less than 0.05 was considered statistically significant. All reported p values are two-sided. Univariate analysis was performed to assess the risk factors for clinical outcome among the study patients. Definitions 1. An episode of bacteremia is defined as the period of 14 days from the time of collection of the first blood culture positive for E. coli or Klebsiella spp. 2. Nosocomial bacteremia: defined as E. coli or Klebsiella spp. bacteremia occurring among patients more than 48 hours after admission to the hospital or among those patients who had an invasive procedure done (minor surgical procedure, intravenous administration of drugs or placement of a urinary catheter) as an outpatient and the bacteremia was attributable to that procedure. 3. Previous antibiotic therapy is defined as antibiotics given

3 SUPPLEMENT TO JAPI december 2010 VOL Fig. 1 : Antibiotic resistance pattern of isolates from bacteremic patients (n=131): ESBL producing vs. non-esbl producing isolates for at least 2 days within the 14 days before an episode of E. coli or Klebsiella spp. bacteremia. 4. Mortality was death from any cause within 14 days from the date of the first positive blood culture for E. coli or Klebsiella spp. 5. An appropriate antibiotic was defined as an antibiotic to which the bacterial isolate was susceptible in-vitro. The study protocol was approved by the institutional review board of Christian Medical College, Vellore, and written informed consent was obtained from all participants. Results One hundred and thirty one episodes of bacteremia were studied during the period of four months from 1 st February 2007 to 31 st May The baseline characteristics are shown in table 1. Seventy eight (59.54%) subjects were males. The most common co-morbid condition was type 2 diabetes, seen in 56 (42.7%) patients. Multiple co-morbidities (2 or more) were seen in 39.7 % (52/131) of patients. The most frequent primary source of the bacteremia was the urinary tract, seen in 59 (45.04%) patients, followed by an intra-abdominal infection in 29 (22.14%), and pneumonia in 7 (5.34%). In 36 (27.48%) patients, the primary source could not be identified ( primary blood-stream infection ). One hundred and two (77.86%) episodes of bacteremia were caused by E. coli and 29 by (22.14%) Klebsiella spp. Eighty two (62.6%) these episodes were nosocomially acquired. A comparison of the co-morbidity profile of patients with nosocomial and community acquired infections revealed that nosocomial infections were more common in patients who had undergone recent surgery (25.6% vs. 4.1%; p=0.002) and in those with solid organ tumors (12.2% vs. 0%; p = 0.011). Antimicrobial susceptibility (Figures 1 and 2): In the preliminary screening test by disc diffusion, 73.5% of E coli and 72.4 % of Klebsiella spp were resistant to cefotaxime and ceftazidime. All these isolates were phenotypically confirmed to be ESBL producers by the double disc diffusion test. Among Fig. 2: Antibiotic resistance pattern of isolates from bacteremic patients (n=131): community acquired vs. nosocomial infections the ESBL producers, 99.2 % were susceptible to carbapenems; 26.7% to 3 rd generation cephalosporins and 7.2% to piperacillintazobactam. Bacteremia due to ESBL producing E. coli and Klebsiella spp were significantly more common in nosocomially acquired infections when compared to community acquired infections (85.37% versus 53.06%; p= <0.001). There was no significant difference in the median age, sex ratio or co-morbidity profile among patients with bacteremia due to ESBL producing and non-esbl producing strains (Table 1). Among this study population, 39 (29.8%) patients had history of antibiotic receipt in the preceding two weeks. The history of antibiotic use was significantly more common among patients who had infections due to ESBL producing strains (p = 0.006). The most common antibiotics used were 3 rd and 4 th generation cephalosporins (N=20; 51.3%) (p=0.017 for comparison between ESBL and non-esbl producing strains). The other classes of antimicrobials used were: beta-lactams with or without betalactamase inhibitors 7 (17.9%), aminoglycosides 15 (38.5%), fluoroquinolones, 13 (33.3%), carbapenems 3 (7.7%) and others 5 (12.8%). Outcome (Table 2): One hundred and twenty two patients were followed up for 14 days from the time of diagnosis of bacteremia. The overall mortality was 23.6% (31 patients). There was no significant difference in mortality among patients with bacteremias due to ESBL and non-esbl producing isolates (p=1.0).we also found no difference in the mortality between nosocomial and community acquired infections (p= 0.52), E. coli and Klebsiella spp. infections (p=1.0), appropriate and inappropriate initial choice of antibiotic (p= 0.54) or the use of carbapenems and other class of antibiotics as initial therapy (p=1.0). Mortality was higher among patients with pneumonia as the primary source of bacteremia (odds ratio, 8.56; 95% confidence intervals ; p=0.01). Discussion ESBL production confers resistance to all the beta-lactam antibiotics, except carbapenems and cephamycins. In addition, ESBL encoding plasmids also carry genes which encode resistance to other class of antibiotics such as fluoroquinolones,

4 16 SUPPLEMENT TO JAPI december 2010 VOL. 58 Characteristic Table 2: Predictors of Mortality Mortality (%) Odds ratio (95% CI) P value Organism E. coli Klebsiella spp ( ) Place of aquisition Nosocomial Community ( ) ESBL production Yes No ( ) Initial antibiotic appropriate Yes No ( ) Carbapenems as initial treatment Yes No ( ) Primary source of infection Urinary tract ( ) Intra abdominal ( ) Undetermined ( ) Pneumonia ( ) 0.01 aminoglycosides and sulfonamides. 8 Thus, limited antibiotic choices are available for the treatment of infections caused by these strains. In these circumstances, it is imperative to quantify the problem and reinforce guidelines promoting appropriate antibiotic use. There are very few reports from India on the prevalence of bacteremia due to ESBL producing organisms among hospitalized patients, their risk factors and treatment outcomes. We prospectively evaluated 131 episodes of bacteremia due to E. coli or Klebsiella spp. occurring among in-patients in a large tertiary care teaching hospital. We chose bacteremic patients as the study population to avoid the difficulty in differentiating infection and colonization in other non-sterile clinical specimens such as sputum, urine, endotracheal aspirate etc. We found that urinary tract infection was the commonest primary source of bacteremia, followed by an intra-abdominal infection. This was in concordance with the findings of other studies. 9 However, in a significant number of patients the source of bacteremia could not be determined. Our study revealed that overall 73.3% of hospitalized bacteremic patients had infections due to ESBL producing strains of E. coli or Klebsiella spp. Other studies from India (which included isolates from all clinical specimens) have reported similar prevalence of ESBL production among Enterobacteriaceae. 1 A study reported from the All India Institute of Medical Sciences, New Delhi, in 2002 showed that 68% of the Enterobacteriaceae were ESBL producing strains. 3 The unusually high prevalence of ESBL producers in the present study was due to the setting of the study (a large tertiary care referral center) and the majority of infections being nosocomially acquired. ESBL production was seen among 85.37% of isolates from nosocomial infections and 53.06% of community acquired infections. The prevalence of ESBL production among isolates from community acquired infections was alarmingly high when compared to other studies. 9 The high prevalence of infections due to community acquired ESBL producing Enterobacteriaceae could have been as result of excessive use of 3 rd generation cephalosporins in the community. Other studies from our institution have shown widely varying prevalence of ESBL producing E. coli among commensals from healthy subjects (27% fecal carriage rate among antibiotic naïve healthy volunteers from a tribal village 10 and 1.6% among urinary commensals of healthy pregnant women. 11 These data indicate that ESBL producing Enterobacteriaceae have become widely prevalent and endemic in a variety of healthcare settings (both in the hospitals and the community) in India. In our study, we found prior antibiotic treatment, especially 3 rd or 4 th generation cephalosporins, the most important risk factor associated with bloodstream infections due to ESBL producing Enterobacteriaceae. Excessive use of 3 rd and 4 th generation cephalosporins, the most commonly used antibiotic class in hospitals, is well known to exert selective pressure for development of resistance, especially ESBL production. Overall, a high degree of resistance to multiple classes of antibiotics was noted, especially to the 3 rd generation cephalosporins (73.3%), beta-lactam and beta-lactamase inhibitor combinations (67.2 to 81.7 %) and fluoroquinolones (73.3%). These results were comparable to those of a study done at The National Institute of Communicable Diseases in New Delhi in 2004, which showed a high frequency of resistance among K. pneumoniae for the cephalosporins and non-cephalosporins. 12 Carbapenems were the most active among all the antimicrobials tested. Only one isolate of E. coli (from a patient with nosocomial urinary tract infection) was resistant to imipenem. After carbapenems, aminoglycosides were found to be the most active class of antibiotics (amikacin resistance 51.9%). The 14-day mortality was %, a finding similar to other studies in bacteremic patients. 9 The initial antibiotic choice was appropriate in 43.75% of the patients with bacteremia due to ESBL producing isolates and in % of those with non-esbl producing isolates (p<0.001). Contrary to previous reports, 9,13 we did not find an association between inappropriate initial antibiotic treatment or ESBL production and mortality. Although not evaluated in randomized controlled trails, carbapenems are considered the most reliable class of antibiotics for treatment of infections caused by ESBL producing Enterobacteriaceae due to its stability against the hydrolytic effects of different beta-lactamases including ESBL. Imipenem was the most active agent in-vitro in our study also; but we found that the use of a carbapenem as the initial antibiotic did not affect the outcome. Our observations most likely indicate the lack of statistical power of this sample size to demonstrate an effect. The mortality among patients with pneumonia was substantially higher than other groups (OR, 8.56; 95% CI, ; p=0.01). In conclusion, our study shows a very high prevalence of ESBL producing Enterobacteriaceae among hospitalized patients with bloodstream infections, even among those patients with community acquired infections. We also noted very high rates of resistance to most classes of antimicrobials, except carbapenems. Therefore, carbapenems should be the initial empiric choice for

5 SUPPLEMENT TO JAPI december 2010 VOL serious, life-threatening infections caused by ESBL producing Enterobacteriaceae, with de-escalation when culture and susceptibility results become available. The empiric use of 3 rd and 4 th generation should be curtailed and stringent infection control measures have to be implemented in hospitals to prevent emergence and further spread of these pathogens. Our study also highlights the need for the continued monitoring of antimicrobial susceptibility patterns of important bacterial pathogens, so that rational antibiotic policies can be formulated. Acknowledgement The authors thank the Christian Medical College Fluid Research Grant for the financial assistance provided for this work. References 1. Jain A, Roy I, Gupta MK, Kumar M, Agarwal SK. Prevalence of extended-spectrum beta-lactamase-producing Gram-negative bacteria in septicaemic neonates in a tertiary care hospital. J Med Microbiol 2003;52: Hansotia JB, Agarwal V, Pathak AA, Saoji AM. Extended spectrum beta-lactamase mediated resistance to third generation cephalosporins in Klebsiella pneumoniae in Nagpur, central India. Indian J Med Res 1997;105: Mathur P, Kapil A, Das B, Dhawan B. Prevalence of extended spectrum beta lactamase producing gram negative bacteria in a tertiary care hospital. Indian J Med Res 2002;115: Chow JW, Yu VL. Combination antibiotic therapy versus monotherapy for gram-negative bacteraemia: a commentary. Int J Antimicrob Agents 1999;11: Garner JS, Jarvis WR, Emori TG, Horan TC, Hughes JM. CDC definitions for nosocomial infections, Am J Infect Control 1988;16: Myers and Koshi s Manual of Diagnostic procedure in Medical Microbiology and Immunology/Serology. Faculty, Department of Clinical Microbiology, Christian Medical College and Hospital, Vellore. India. Pondicherry, All India Press, Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing; Approved standards M2- A7, eighteenth informational supplement. CLSI document M100 S 18. Wayne, PA Medeiros AA. Evolution and dissemination of beta-lactamases accelerated by generations of beta-lactam antibiotics. Clin Infect Dis 1997;24Suppl1:S Paterson DL, Hujer KM, Hujer AM, Yeiser B, Bonomo MD, Rice LB, et al. Extended-spectrum beta-lactamases in Klebsiella pneumoniae bloodstream isolates from seven countries: dominance and widespread prevalence of SHV- and CTX-M-type beta-lactamases. Antimicrob Agents Chemother 2003;47: Mathai D. Clinico-Epidemiologic and Molecular Characterization of MRSA and ESBL Producing Escherichia coli, Klebsiella spp. and Enterobacter spp., Causing Hospital and Community - Acquired Infections in Indian Hospitals. Ph.D dissertation submitted to TN Dr.MGR Medical University Mathai E, Chandy S, Thomas K, Antoniswamy B, Joseph I, Mathai M, et al. Antimicrobial resistance surveillance among commensal Escherichia coli in rural and urban areas in Southern India. Trop Med Int Health 2008;13: Grover SS, Sharma M, Pasha ST, Singh G, Lal S. Antimicrobial susceptibility pattern and prevalence of extended spectrum betalactamase (ESBLs) producing strains of Klebsiella pneumoniae from a major hospital in New Delhi. J Commun Dis 2004;36: Du B, Long Y, Liu H, Chen D, Liu D, Xu Y, et al. Extendedspectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae bloodstream infection: risk factors and clinical outcome. Intensive Care Med 2002;28: B o o k R e v i e w Hospital Acquired Infections Prevention and Control Purva Mathur Infection Control has become a major focus in the study of Infectious Diseases. It is of paramount importance today for every health care facility, however the essential elements of this topic are all scattered in the literature. This book brings together all this information along with pearls of clinical wisdom & thus is a thorough guide on Infection Control. Each topic is dealt with in great detail, incorporating recommendations from recent guidelines (World Health Organization and Centres for Disease Control). The author has included every topic that is relevant to infection control, from setting up an infection control committee to prevention of infection in operating theatres, to dealing with construction work in a hospital site. The layout of the chapters is very user friendly, with an outline at the beginning of each chapter. The author has simplified the information presented with boxes, tables, pictures and diagrams. The chapters are very well referenced and complete. There are few books on this subject that are so well written and we would like to congratulate the author on her commendable effort. The book is a valuable resource for clinicians, microbiologists, nurses, ancillary staff & hospital administrators. We would suggest that the book be introduced into the medical curriculum so that the principles of infection control are inculcated in our young doctors. The book has demystified infection control and can be used all health-care professionals. It should find a place wherever infection control is to be practiced, in short, everywhere. Dr. Rajeev Soman, Consultant Physician, Dr. Anjali Shetty, Consultant Microbiologist, PD Hinduja Hospital, Mumbai