Antimicrobial resistance among uropathogens with reference to extended spectrum β-lactamase production

Abstract

Emerging antibiotic resistance due to extended spectrum β-lactamases (ESBLs) production limited the use of β-lactam antibiotics against uropathogens. The current prospective study was conducted in the Department of Microbiology, Yuvaraja’s College, Mysore, India from December 2010 to December 2013 by collecting samples from K.R. Hospital and C.S.I. Holdsworth Memorial Hospital, Mysore. Three hundred urine specimen were collected from the symptomatic patient of urinary tract infection (UTI) and study continued further with an aim to determine the prevalence of UTI, the effect of gender and age on its prevalence, microscopic findings, profile of etiological uropathogens, their antimicrobial resistance pattern, further detection of extended spectrum β-lactamases (ESBLs) and containment of spread of drug resistant bacteria in hospital. In the present study, 160 (53.4%) of the urine specimen were received from females and remaining 140 (46.6%) from males. Microscopic examination of the urine specimen revealed pyuria (pus cells >10 HPF-1) in 50.0% of the case and hematuria in (RBCs >10 HPF-1) in 10.0% (P < 0.000, χ2 42.000). Detection of pyuria gives the early presumptive diagnosis of UTI, which helps to start the empirical treatment early. The urine culture study revealed significant bacteriuria in 180 (60%) (P < 0.001, χ212.000); among them 91 (50.5%) were from outpatient department and 89 (49.5%) from inpatient department. In the present study, the reason for high prevalence of UTI could be due to the selection of only symptomatic patients. Because of the short urethra, close proximity of the female urethral meatus to anus, heavy periurethral cutaneous colonization, sexual intercourse and poor personal hygiene influences the higher prevalence of UTI in females, which proves true in the present study where majority of significant bacteriuria 111 (61.7%) were seen in females and remaining 69 (38.3%) were seen in males. With advance of age in male due to prostatic enlargement and neurogenic bladder chances of acquitting UTI is high, which proves true in the present study where majority (75.4%) of significant bacteriuria in males was seen in higher age group i.e., >50 years. The scenario is just reverse in the females. In females majority (83.8%) of the significant bacteriuria cases were seen in age group 11-50 years. This findings indicates that in females UTI is much common problem in younger age compared to older age group >50 years. This might be due to several xiii factors but the most important one is that females are highly sexually active during reproductive phase of life which falls within this range of age. Among significant bacteriuria, 165 (91.7%) were having growth of single species of bacteria, whereas 15 (8.3%) yielded the growth of two different bacterial species (P < 0.000, χ2125.000). Gram negative bacilli (GNB) posses several virulence factors responsible for their attachment to uroepithelium. They colonize the urogenital mucosa with adhesins, pili and fimbriae, this enhances the probability of UTI, which is true in the present study, among 195 isolated uropathogens, 180 (92.3%) were GNB and 15 (7.7%) were gram positive cocci (GPC) (P < 0.000, χ2139.615). Bacteriological profile of uropathogens revealed Escherichia coli, 110 (56.4%) as the leading pathogen; followed by Klebsiella pneumoniae, 30 (15.4%); K. oxitoca, 10 (5.1%); Pseudomonas aeruginosa, 10 (5.1%); Proteus mirabilis, 6 (3.1%); Enterobacter aerogenes, 6 (3.1%); Citrobacter freundii, 4 (2.1%); Acinetobacter anitratus, 4 (2.1%); Staphylococcus aureus, 6 (3.1%); S. saprophyticus, 4 (2.1%) and Enterococcus faecalis, 5 (2.6%) (P < 0.000, χ2 559.826). The overall resistance pattern of uropathogens were 195 (100.0%) for ampicillin, 152 (78.0%) for nalidixic acid, 148 (75.8%) for cefixime, 141 (72.3%) for norfloxacin, 139 (71.3%) for cotrimoxazole, 135 (69.2%) for ciprofloxacin, 134 (68.7%) for amoxicillin/clavulanic acid, 123 (63.1%) for ceftazidime, 113 (58.0%) for aztreonam and ceftriaxone, 105 (53.8%) for cefotaxime and cefpodoxime, 83 (42.6%) for gentamicin, 72 (36.9%) nitrofurantoin, 63 (32.3%) for amikacin, 10 (5.1%) for cefoxitin and imipenem and 9 (4.6%) for meropenem. The resistance pattern of ESBLs producing E. coli were 95% for amoxicillin/clavulanic acid and cotrimoxazole, 91.7% for nalidixic acid, 90.0% for cefotaxime and cefpodoxime, 88.7% for norfloxacin, 86.7% for ciprofloxacin, 83.3% for cefixime and ceftazidime, 76.7% for aztreonam and ceftriaxone, 63.3% for gentamicin, 60.0% for nitrofurantoin, 41.7% for amikacin, 10.0% for imipenem and 8.3% for meropenem. The resistance pattern of non-ESBL producer E. coli were 66% for nalidixic acid, 60.0% for norfloxacin, 54.0% for cefixime, 52.0% for amoxicillin/clavulanic acid and cotrimoxazole, 50.0% for ciprofloxacin, 44.0% for ceftazidime, 40.0% for aztreonam and ceftriaxone, 16.0% for nitrofurantoin, 12.0% for cefotaxime, cefpodoxime and gentamicin, 4.0% for amikacin and 0.0% for imipenem and meropenem. The resistance pattern of ESBLs producer K. pneumoniae xiv were 94.7% for nalidixic acid, 89.5% for norfloxacin, 84.2% for cefixime, ceftazidime and ciprofloxacin, 79.0% for amoxicillin/clavulanic acid, aztreonam, cefpodoxime, ceftriaxone, cefotaxime and cotrimoxazole, 63.3% for nitrofurantoin, 57.9% for gentamicin, 42.1% for amikacin, 15.8% for cefoxitin and imipenem and 10.5% for meropenem. The resistance pattern of non-ESBLs producer K. pneumoniae were 63.6% for nalidixic acid, 54.6% for amoxicillin/clavulanic acid and norfloxacin, 45.4% for cefixime, 27.3% for ciprofloxacin, 18.2% for aztreonam, cefotaxime, cefpodoxime, ceftriaxone and cotrimoxazole, 18.2% for amikacin, nitrofurantoin and gentamicin, 0.0% for cefoxitin, imipenem and meropenem. The resistance pattern of ESBLs producer K. oxitoca was 80.0% for ciprofloxacin, cotrimoxazole, nalidixic acid and norfloxacin, followed by 60.0% for amoxicillin/clavulanic acid, ceftriaxone, aztreonam, cefpodoxime, cefotaxime, cefixime, ceftazidime and gentamicin, 40.0% for amikacin and nitrofurantoin, 0.0% for cefoxitin and imipenem and 20.0% meropenem. The resistance pattern of non-ESBLs producer K. oxitoca were 60.0% for amoxicillin/clavulanic acid, aztreonam, cefixime, cefotaxime, cefpodoxime, ceftazidime, ceftriaxone, ciprofloxacin, cotrimoxazole, gentamicin, nalidixic acid and norfloxacin, followed by 40.0% for amikacin, nitrofurantoin, 0.0% for cefoxitin, imipenem and meropenem. The resistance pattern of ESBL producer P. aeruginosa were 100.0% for cotrimoxazole and nalidixic acid, followed by 75.0% for amoxicillin/clavulanic acid, aztreonam, cefixime, ceftriaxone, ceftazidime, ciprofloxacin, and norfloxacin, 50% for amikacin, cefpodoxime, cefotaxime, gentamicin and nitrofurantoin, 25.0% for cefoxitin, imipenem and meropenem. The resistance pattern of non-ESBL producer P. aeruginosa were 66.7% for ciprofloxacin, cotrimoxazole, nalidixic acid and norfloxacin, followed by 50.0% for amoxicillin/clavulanic acid, aztreonam, cefixime, cefpodoxime, cefotaxime, ceftriaxone, ceftazidime, 33.3% for gentamicin and nitrofurantoin, (16.7%) for amikacin, 0.0% for cefoxitin, imipenem and meropenem. ESBLs producer gram negative uropathogen were highly resistant to almost all the antibiotics tested (amikacin, amoxicillin/clavulanic acid, aztreonam, cefixime, cotrimoxazole, ciprofloxacin, ceftazidime, ceftriaxone, cefotaxime, cefpodoxime, gentamicin, nalidixic acid, nitrofurantoin and norfloxacin) in comparison to non-ESBL producer, where the range was as follows: 41.0-95.0% vs 40.0-66.0% for E. coli, 42.1- 94.7% vs 18.2- 63.6% for K. pneumoniae, 40.0-80.0% vs 40.0-60.0% for K. oxitoca and 25.0- xv 100.0% vs 16.7-66.7% for P. aeruginosa. The range of resistance for cefoxitin, imipenem and meropenem among ESBLs producer and non-ESBLs producer uropathogenic isolates was as follows, 8.3-10% vs 0.0% for E. coli, 10.5-15.8% vs 0.0% for K. pneumoniae and 0.0-20.0% vs 0.0% for K. oxitoca. In the present study, the phenotypic detection of ESBLs was done by double disc synergy test (DDST), Clinical and laboratory standards institute (CLSI) confirmatory test and epsilometer test (E-test) method. Among 180 gram negative uropathogens tested for ESBLs production by the phenotypic method 96 (53.3%) was positive. Among E. coli ESBLs production was seen in 60 (54.55%) of the isolates by DDST and CLSI confirmatory test (P >0.340, χ2 0.909), whereas by E-test ESBL production was detected in 61(55.45%) (P > 0.253, χ2 1.309). Among K. pneumoniae ESBL production was seen in 19 (63.3%) of the isolates by the phenotypic method (P > 0.144, χ2 2.133), whereas 50.0% of the K. oxitoca, P. mirabilis and C. freundii; (40.0%) of the P. aeruginosa, 33.3% of the E. aerogenes and 25.0% of the A. anitratus was positive for ESBL production. Polymerase chain reaction (PCR) study revealed 54 (90.0%) positive for ESBLs gene among E. coli (P < 0.000, χ2 38.400); where blaCTX-M 36 (66.7%) was leading followed by blaSHV 10 (18.5%) and blaTEM 8 (14.8%) (P < 0.000, χ2 27.111); 17 (89.5%) positive for ESBLs gene among K. pneumoniae (P < 0.001, χ211.842); where blaCTX-M ESBLs 10 (58.8%) was the leading followed by blaSHV 6 (35.3%) and blaTEM 1 (5.9%) (P < 0.028, χ2 7.176); and 4 (80.0%) positive for ESBLs gene among K. oxitoca, where blaCTX-M ESBLs 2 (50.0%) was the leading followed by blaSHV 1 (25.0%) and blaTEM 1 (25.0%). Out of 100 collected swab from hands of health care personnel and patients, bacterial growth recovered from 60 which comprises six different bacterial genera, among them Staphylococcus sp. 58.3% was the leading one, followed by P. aeruginosa 15.0%, E. coli and E. faecalis of 8.3%, Klebsiella sp. and Acinetobacter sp. of 5.0%. The resistance pattern exhibited by pathogen isolated from hands swab was 40.0% for amikacin, 56.7% for amoxicillin/clavulanic acid, 60.0% for cefixime, 45.0% for cefotaxime, 48.3% for ceftazidime, 45.0% for ceftriaxone, 55.0% for ciprofloxacin, 66.7% for cotrimoxazole, 68.3% for nalidixic acid, 65.0% for norfloxacin, 46.7% for nitrofurantoin, 45.0% for gentamicin, 6.7% for imipenem and 5.0% for meropenem. The study revealed 36.0% of the respondent harbours S. aureus in nasal cavity, among them 16% were methicillin resistant S. aureus (MRSA) and remaining methicillin sensitive S. aureus xvi (MSSA). The distribution of organism isolated from swab specimen collected from various articles and surface of hospital revealed P. aeruginosa 23.1% as a leading pathogen, followed by S. aureus and E. coli 16.2% each, coagulase negative S. aureus 13.9%, E. faecalis 10.6%, K. pneumoniae 9.2%, Acinetobacter sp. 7.0%, Enterobacter sp. 2.2% and Proteus sp. 1.8%. The mean antimicrobial resistance pattern exhibited by the pathogens isolated from swab specimen collected from various articles and surface of hospital was 42.5% for amikacin, 60.2% for amoxicillin/clavulanic acid, 63.8% for cefixime, 53.0% for cefotaxime, 54.4% for ceftazidime, 54.4% for ceftriaxone, 65.1% for ciprofloxacin, 73.0% for cotrimoxazole, 75.0% for nalidixic acid, 66.5% for norfloxacin, 45.6% for nitrofurantoin, 46.0% for gentamycin, 6.5% for imipenem and 6.0% for meropenem. The assessment of knowledge, attitudes and practices pertaining to prevention of infection control to prevent the spread of multi drug resistant bacteria among different categories of health care personnel showed that doctors had better knowledge-attitude-practice regarding infection control, followed by nurses/ paramedical staff; whereas ward aides have very poor knowledge-attitudepractice for the same. The rise and gradual drop in the scores seen among various categories of health care personnel. The impact of education was similar in all categories of health care personnel. So the present study recommends regular training of health care personnel especially ward aides regarding infection control. Monitoring of ESBLs production and antimicrobial susceptibility testing are necessary to avoid treatment failure. Enhanced infection control programme is necessary to control the spread of multidrug resistant bacteria in hospital.