European Journal of Cardiovascular Medicine

Urinary tract infections are one of the most commonly encountered problems in outpatient departments. It is caused by abnormal growth of pathogens which can present as fever, dysuria and lower abdominal pain and can lead to serious consequences such as nephritis, scarring of the kidney [1,2]. UTI is common in young women of 14-24 years old, the prevalence of UTIs increases with age [3]. Between 50% to 60% of adult women will have at least one UTI in their life, and close to 10% of post menopausal women indicate that they had a UTI in the previous year [4]. The pathogens causing UTI usually colonize the urethra or periurethral space after being transmitted into the urinary system. They migrate into the bladder and kidney, resulting in inflammatory response. Transmission of bacteria usually from gastrointestinal tract of patients own flora or from exogenous source. Most common organisms causing UTI are Esch.coli and Klebsiella pneumoniae which are most common commensals of GIT.

Antibiotic resistance is an emerging global problem. Epidemics of antibiotic susceptibility patterns of pathogens are changing. The incidence of multidrug resistance in pathogenic and opportunistic bacteria has been increasing in recent years. MDR Pseudomonas aeruginosa, ESBL Enterobacteriaceae were also frequently cultured [5]. Misuse and injudicious use of antibiotics and empirical treatment have been the major contributory factors in the emergence of MDR bacteria. For Instance, the prevalence of MDR Escherichia coli among outpatient isolates in the United states increased from 9% in 2001 to 17% in 2010 [6].

Diagnosis of UTI is usually made by urine routine examination and urine cultures. These diagnostic tests are indicated for both complicated, uncomplicated UTIs and asymptomatic bacteriuria. More than the detection of bacteria during urine analysis, detection of MDR pathogens and their AST pattern plays a vital role to treat the patient accordingly.

Here in this research work we would like to project the data clinicians about ESBLs pathogens and its susceptibility pattern. It will aid in the recommendations of empiric therapy to UTI patients in this community.

Study Settings:

A Prospective observational study on a total of 500 culture positive urinary samples was conducted at department of Microbiology, Government Medical College, Anantapur, after taking consent from the study population. All the samples were studied to detect ESBL pathogens.

Collection of samples:

Regular hospital rounds to find the cases of urinary tract infections (UTI) helped us to collect the appropriate relevant urine samples from the patients suffering from UTI. All the patients were instructed to collect midstream clean catch urine samples after thorough cleaning of genitals.

Inclusion criteria: All inpatient and outpatient samples of UTI patients of all ages and both sexes.

Exclusion criteria: All clinical urine samples for routine examination were excluded.

Processing of samples:

All urinary samples were transported to the Department of Microbiology immediately under suitable conditions. Specimens were cultured on nutrient agar, CLED agar as a semi quantitative method and these plates were incubated for 24 hours at 370C. Growth plates were further analyzed by gram stain, biochemical reactions to identify the pathogen. Modified Kirby Bauer method was followed to do antibiotic sensitivity testing on Mueller Hinton agar (MHA) plates.

ESBL Detection:

All gram negative bacterial isolates were further evaluated for ESBL detection by using combined disc diffusion test (CDDT) and double disc synergy test (DDST).

Combined Disc Diffusion Test: A combination of cefotaxime+clavulanic acid (30/10µg) and cefotaxime (30 µg) was used. Both discs were placed on Muller Hinton agar at a distance of 20 mm on agar plate and incubated for 24 hours at 370C. A zone diameter of >=5mm in CTX/CL versus CTX had been considered positive for ESBL producers.

Double disc synergy test: A combination of Amoxyclav (20/10µg), Aztreonam (30µg), ceftriaxone (30µg) and ceftazidime (30µg)and cefotaxime (30µg). At center an amoxicillin-clavulanic acid disc was placed and these discs were placed at a distance of 15mm from center to center. Development of the zone of inhibition towards the clavulanate disc at 370C after 24 hours incubation was indicative of a potential ESBL positive organism.

Data Collection and analysis:

All the patient details were collected in a spread excel sheet along with culture and ST details. Results were tabulated and presented as numbers and percentages.

A total of 500 culture positive urinary isolates derived from UTI patients were evaluated under this study. In this study 273 (54.6%) gram negative isolates, 193 (38.6%) gram positive isolates and 34 (6.8%) candida isolates were identified. Among the urinary pathogenic isolates, Esch.coli (22.85) was the predominant pathogen followed by S.aureus (19.2%), Klebsiella species (11.6%) (Fig 1). Around 40% of isolates were detected in the 12-30 years age group population. Females were predominantly affected with UTI (Table 1).

Bacteria are the major causative organisms responsible for more than 95% of UTI cases. Now-a-days, treating an infection is becoming difficult due to decreasing the pipeline of antibiotics invention and increasing the resistance to antibiotics. There is a growing concern for multidrug-resistant gram negative bacteria which produce ESBLs [5]. ESBLs are class A β-lactamases that hydrolyse penicillin, oxyimino-cephalosporins, and monobactams but not cephamycins or carbapenems [7]. They are inhibited in vitro by clavulanate. ESBLs are often plasmid – mediated enzymes and have various important types including VEB, PER, BEL-1, BES-1, SFO-1, TLA and IBC [8].

Around 40% of isolates were detected in the 12-30 years age group population. Females were predominantly affected with UTI in the present study. The prevalence rate of ESBL UTIs in relation to age groups varies. Few studies noted the UTI in children  [9] and few other  research works noted in elderly age group  [10]. In most of the studies females are affected by UTIs predominantly [11-13]. Variation of prevalence rate in terms of age and sex could be due to differences in climatic conditions, hygienic practices and antibiotic usage from community to community.

Among the urinary pathogenic isolates, Esch.coli (22.85) was the predominant pathogen followed by S.aureus (19.2%), Klebsiella species (11.6%). ESBL producers include 62.85% of Escherichia coli, 35.71% of Klebsiella and 1.4% of Proteus mirabilis isolates. Escherichia coli is the most common pathogen causing UTI in both hospital and community settings. Escherichia coli is a commensal of GIT, which can easily access into the genitourinary tract, it adheres to urethra with its pili which is the foremost virulence factor causing infection. Usually the 1% of MDR is high in hospital acquired infections when compared to community acquired infections, might be because of exchange of resistance genes within the bacterial population and transmission of MDR pathogens from one patient to another patient either by direct or indirect transmission. A Study from Turkey observed 50.5% of prevalence of ESBL-E.coli-UTI hospital acquired, whereas community-acquired ESBL-E.coli-UTI had a prevalence rate of 38.2% [14].

A Study in Qatar on ESBL-UTI found that the prevalence among the adult population was 25.2%. Most common organisms isolated in UTI and ESBL detection were also high in Escherichia coli (54.5%) and Klebsiella species (16.5%) [10]. Many other studies noted Escherichia coli was the predominant pathogen in ESBL-UTI, it was 26.8%, 52%, 62% in Qatar [15], Syria [16], and Jordan [17]  respectively.

A study conducted in the costal part of Andhra Pradesh [18] showed that ESBL producers are higher in in-patients than out-patients, fecal carriage of these ESBL producing organisms was found to be higher among in-patients (42.03%) followed by out-patients (29.37%), healthy patients (9.72%) predominant pathogen detected in their study was Escherichia coli (55.17%) in similar to our study. A Study conducted in Mumbai at a tertiary care hospital observed prevalence of ESBLs producing Esch.coli (34.85) were increased than Klebsiella species (30.7%) [19].

ESBL producers showed highest sensitivity to amikacin, imipenem, colistin and polymyxin B and the poorest sensitivity to cefotaxime and amoxyclav. In line with this study other studies also reported a similar susceptibility pattern [11,20,21].

In the present study the DDST  and CDT  proved as best methods with overall percent agreement of 99.2%. Shiferaw Teklu D et al [22] observed the overall percent agreement between DDST and CDT as 91.7% and the PPA and NPA was 91.0% and 100% respectively. Hooja S and his colleagues identified 60.2% by CDT and 56.8% by DDST [23] and Giriyapur RS and his colleagues identified 63.89% by CDT and 56.23% by DDST as ESBLs producers [24].

We conclude that most common pathogens were Esch.coli, S.aureus and Klebsiella species. 38.5% of Escherichia coli isolate and 43.10% of Klebsiella isolates were predominant ESBL producers. Majority of the ESBL producers were sensitive to Imipenem, meropenem, amikacin, nitrofurantoin and colistin. Active screening of UTI suspected patients for ESBLs colonization is important to initiate appropriate infection control measures that includes cohorting, de-escalating the antibiotics and confined to narrow spectrum antibiotics, and adherence to hospital infection control protocols.

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