1. INTRODUCTION Infectious disease is a clinically evident condition that results from a pathogenic agent which includes bacteria, fungi, virus, protozoa, multi-cellular parasites and aberrant proteins known as prions. Infectious pathologies (pathogens) are usually contagious diseases (also known as communicable diseases) due to their potential transmission from one species to another (Dorlands, 2007). The transmission of an infectious disease may occur through one or more diverse pathways that include physical contacts with infected person, through liquid, food, body fluids, contaminated objects, airborne inhalation or through vector (McGraw, 2005). Infectious diseases are the second largest cause of mortality in the world accounting for more than 13 million deaths annually (Cohen, 2000). In addition to causing deaths these diseases also disable millions of adults, diminishing their quality of life, decreasing productivity, and creating financial hardships for families (Breman, et al., 2004; UNAIDS and WHO, 2007). Many diseases are curable, others are manageable with treatment, and many are preventable with vaccines and other tools. However, getting these interventions to the people in order to prevent or control the disease has been challenging. Urinary tract infection (UTI) is one among them. UTI is the most common bacterial infectious disease in the community practice with a high rate of morbidity and financial cost. It has been estimated that 150 million people were infected with UTI per annum worldwide which cost global economy more than 6 billion US dollars (Stamm, 1993). Bacterial infections causing UTI can be symptomatic or asymptomatic. The symptomatic urinary tract infection can be uncomplicated or complicated. Uncomplicated symptomatic uinary tract infection is characterized by frequency, urgency, dysuria, or supra pubic pain in a woman with a normal genitourinary tract (Sampson, et al., 1999; Hooton, et al., 1997). Complicated symptomatic urinary infection in women is with functional or structural abnormalities of the genitourinary tract which involve either the bladder (cystitis) or kidneys (pyelonepritis) (Nicolle, 2001). The asymptomatic urinary tract infection is defined as a persistent, actively multiplying bacteria within the urinary tract without any symptoms of infection (Uncu, et al., 2002). 1
UTI can affect lower and sometimes both lower and upper urinary tracts. The term cystitis has been used to define the lower UTI infection and is characterized by symptoms such as dysuria, frequency, urgency, and suprapubic tenderness. The presence of the lower UTI symptoms does not exclude the upper UTI, which is often present in most of the complicated UTI cases (Sobel, et al., 2010). The treatment of UTI can be classified into uncomplicated and complicated on the basis of their choice of treatment (Sabra, et al., 2012). UTI is more common in females than in males, since the female urethra is structurally less effective for preventing the bacterial entry (Warren, et al., 1999). It may be due to the proximity of the genital tract and urethra (Schaeffer, et al., 2001) and adherence of urothelial mucosa to the mucopolysaccharide lining (Akortha, 2008). The other main factors which make females more prone to UTI are pregnancy and sexual activity (Arul, et al., 2012). In pregnancy, the physiological increase in plasma volume and decrease in urine concentration develop glycosuria in upto 70% women which ultimately leads to bacterial growth in urine (Lucas, et al., 1993). Also in the non pregnant state the uterus is situated over the bladder whereas, in the pregnant state the enlarged uterus affects the urinary tract (Warren, et al., 1982). Sexual activity in females also increases the risk of urethra contamination as the bacteria could be pushed into the urethra during sexual intercourse as well as bacteria being massaged up the urethra into the bladder during child birth (Ebie, et al., 2001: Kolawole, et al., 2009). The use of contraception, such as diaphragms also causes UTI as it pushes against the urethra and makes the urethra unable to empty the bladder completely and the small concentration of urine left in the bladder leads to the growth of bacteria which ultimately causes UTI (Okonko, et al., 2009). The prevalence is also markedly increased if women present certain pre-existing medical conditions, such as diabetes mellitus, sickle cell disease, immuno-deficiency states, urinary tract anatomic anomalies, spinal cord injuries and psychiatric illnesses (Ovalle, et al., 1989). During gestation, untreated UTIs can lead to several pregnancy complications, such as the low birth weight infants, premature delivery, and occasionally, stillbirth (Hill, et al., 2005). Prompt and efficacious treatment of symptomatic UTIs is warranted in pregnant women. Nevertheless, there is still some controversy regarding the screening and treatment of asymptomatic forms during gestation of 2
pregnancy (Lin and Brown, 2010; Lumbiganon, et al., 2010; Schmiemann, et al., 2010). The presence of ASB in pregnancy places patients at an increased risk for the development of cystitis and pyelonephritis with their respective morbidities. A critical meta-analysis by Romero and colleagues (1989) showed the relationship between ASB alone and preterm delivery and low birth weight infants. The risk of preterm delivery in women who had ASB during gestation was two fold greater than those who were never affected (Pfau, et al., 1992). Without treatment, ASB progresses to pyelonephritis in 20% to 40% of pregnant women. In contrast, progression to pyelonephritis in nonpregnant women is only 1% to 2%. Furthermore, the incidence of pyelonephritis in pregnant women without ASB complicating early pregnancy is less than 1%. With appropriate treatment in pregnancy, progression to pyelonephritis can be decreased to 3% (Wing, 1998). Bacteremia occurs in 15 to 20% of cases of pyelonephritis, the most common pathogen is E. coli. Endotoxin of Gram negative bacteria damages the capillary endothelium, diminished vascular resistance, and changes in cardiovascular output. When the active component of endotoxin lipid-a is released into the maternal circulation, it precipitates as a cascade response of proinflammatory cytokines, histamine, and bradykinins that may lead to the more serious complications of septic shock, disseminated intravascular coagulation, respiratory insufficiency, and adult respiratory distress syndrome (ARDS). Pyelonephritis is the most common cause of septic shock in pregnancy (Mabie, et al., 1997). The causative organisms that are isolated in asymptomatic Bacteriuria (ASB), cystitis, and pyelonephritis are similar in pregnant as well as in nonpregnant women. Most of the members of family Enterobacteriaceae encompass most of the colonizing organisms. Escherichia coli are the primary pathogen found in 80 to 90% of initial UTIs and 70 to 80% of recurrent infections. Other gram-negative pathogens include Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, Citrobacter sps, Enterobacter aerogenes, Pseudomonas aeruginosa and of the gram-positive organisms, Streptococcus agalacticae and Staphylococcus saphrophyticus (Ovalle, et al., 2001; Gilstrap, et al., 2001; Foxman, et al., 2003; Hooton, et al., 2003). Antibiotic resistance is now a linked global problem. Dispersion of the successful clones of multidrug resistant (MDR) bacteria is found to be common, the 3
importance of plasmids carrying multiple drug resistance (MDR) markers in Shigella sps and Escherichia coli was first described in the seminal work by Watanabe in Japan over 50 years ago (Watanabe, 1963). These plasmids are capable of self transfer (conjugation) between strains and species and have a mosaic structure that has arisen by recombination and transposition, which is responsible for the capture of different resistance genes, giving rise to the MDR phenotype (Leplae, 2006). Usually antibiotics are given empirically before the laboratory results of urine culture are available to ensure appropriate therapy. However, resistance to the commonly prescribed antibiotics is an expanding global health problem and various studies clearly demonstrate the increasing antibiotic resistance in uropathogens causing both community and nosocomially acquired UTIs (Tenvor and Hughes, 1996; Rahal, et al., 1997; Finch, 1998). Antimicrobial resistance among the Enterobacteriaceae, a group of highly pathogenic Gram negative organisms has increased dramatically in recent years against commonly used antimicrobials such as the tetracyclines, β-lactams, fluoroquinolones, aminoglycosides and co trimoxazole (Fritsch, et al., 2005). Therefore, it is recommended for physicians to obtain information about the local antibiotic resistant rate as stated by the Infectious Disease Society of America (Warren, et al., 1999). In addition, surveillance at the institutional and regional level is required to monitor the changes in susceptibility of uropathogens. Current knowledge of the organisms that cause urinary tract infection is mandatory (Gruneberg, 1982) and the detection of antibiotic resistance and ESβL producer may facilitate the implementation of effective therapy and control measures. Infections due to ESβL producers range from uncomplicated UTI to life threatening sepsis. It is well recognised that there can be marked variation in the incidence and or genotype of the ESβL s in hospitals close to one another, and certainly among countries (Bonnet, 2004). The ESβL enzymes are commonly found in the members of Enterobacteriaceae family and are of over 120 types. Extended-spectrum β-lactamases (ESβLs) are generally acquired by horizontal gene transfer and confer resistance to oxyiminocephalosporins, some being mutant derivatives of established plasmid-mediated β- lactamases (e.g. TEM/SHV) or mobilized from environmental bacteria (e.g. CTX- M) and these enzymes hydrolyze penicillins, broad-spectrum cephalosporins. and 4
monobactams. However, they do not affect cephamycins and carbapenems, and they are inhibited by clavulanic acid (Paterson et al., 2005). Currently, the emergence and rapid dissemination of CTX-M positive ESβL producing bacteria have caused a change in ESβL epidemiology (Paterson, et al., 2005; Livermore, et al., 2005; Pitout, et al., 2008). As these ESβL producing strains often exhibit a multidrug-resistance phenotype, the increase in community-acquired urinary tract infections (UTIs) caused by CTX-M ESβL producers poses significant challenges for the empiric treatment of these infections in the outpatient setting. Furthermore, the recent identification of ESβL producing isolates that have acquired carbapenemases has further limited the therapeutic options available for treatment of these multidrug resistant microorganisms (Nordmann, et al., 2011). The recognition of the importance of ESβL s as a major mechanism of β-lactam resistance throughout the region came with presentation of data from the 1998 1999 SENTRY antimicrobial surveillance programme (Bell, et al., 2002). The incidence of ESβL production (no genotyping was undertaken) among E. coli isolates in the four Chinese sites varied from 13% to 35%. Rates >20% for the ESβL phenotype in Klebsiella pneumoniae in all participating mainland Chinese centres (one reaching 60%), in one each of three Japanese and Taiwanese centres, and in the single Singapore centre and Philippines centre, were confirmed. Such high rates had previously been reported only from South America, in a follow-up study (1998 2002) lower rates were found in K. pneumoniae isolates from Australia and Japan (<10%), but that in China was 30% (Hirakata, 2005). The other area of Asia that is the Indian subcontinent high rates of ESβL production has been reported. One study reported a 68% prevalence of ESβL phenotypes among E. coli and K. pneumoniae isolates, one of the highest rates reported for any country worldwide (Mathai, et al., 2002). A number of other studies in India reported the incidence of ESβL producers to be 6.6 to 68%. In south India, Subha, et al., (2002) reported 6.6% ESβL producers whereas Babypadmini, et al., (2004) reported 40.3%. The ESβL production which was reported among gram negative bacteria by Mathur, et al., (2002) was 68% and Singhal, et al., (2005) detected ESβL in 64% isolates and Rodrigues, et al., (2004) reported 53% ESβL production. Other studies in India have also reported a very high prevalence of ESβL producing Enterobacteriaceae. Accordingly in North India 46% of uropathogens belonging to Enterobacteriaceae were found to be 5
ESβL producers (Akram, et al., 2007). Some reports have also described the molecular epidemiology of the ESβL producers (Ensor, et al., 2006; Lal, et al., 2007; Walsh, et al., 2007). One of the reasons contributing to the high prevalence of ESβL producers in India may be the crowded hospital conditions, including implementation of optimal hygienic practises, likely fuelled by unrestricted use of antimicrobials without doctor s prescription (Ray, et al., 2003). A recent study reported that the ESβL producing Enterobacteriaceae family were responsible for the onset of community infections in India (Gupta and Datta, 2007). CTX-M -15 is known to be having a peculiar association with the community onset of E. coli infections (Nicholas Chanoine, et al., 2008). It may therefore be speculated that, CTX-M-15 producing E. coli has already established in Indian community. The emergence of metallo-β-lactamases (MβLs) with activity against carbapenems (e.g. the VIM and IMP families of enzymes) has compromised the clinical utility of this class of antibiotics (Hawkey, et al., 2007; Walsh, 2008). Resistance to carbapenems may also be induced as a result of increased production of either AmpC or ESβL, coupled with a decrease in porin production or increased efflux (Walsh, 2008; Mena, et al., 2006). In India, the high rates of ESβL producers have recently increased the usage of carbapenem antibiotics, which may provide a selective pressure for the spread of strains producing carbapenemases in the near future. Though the presence of β-lactamases in Enterobacteriaceae have been reported in India, information on the molecular types of ESβL and MβL producers in uropathogens and that too in pregnant women from Karnataka is limited. Population of India standing at 1.2 billion, it surely represents the largest reservoirs of CTX-M ESβL genes in the world. ESβL producing Enterobacteriaceae in India are mostly detected by phenotypic methods. The present study was undertaken to identify the etiology of the uropathogens, prevalence of UTI and their antibiotic resistance profiles, incidence of β-lactamases produing uropathogens and ESβL genes from the isolates of urine samples collected from the pregnant women with suspected UTI from different parts of Karnataka. 6