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1.  Hemagglutination and biofilm formation as virulence markers of uropathogenic Escherichia coli in acute urinary tract infections and urolithiasis 
Urinary tract infections (UTI) are a major public health concern in developing countries. Most UTIs are caused by E. coli, accounting for up to 90% of community-acquired UTIs (CAUTI). Recurrent UTI is considered as a major risk factor for urolithiasis. Virulence factors like adhesins and biofilm have been extensively studied by authors on UPEC isolated from recurrent UTI. The studies on isolates from infection stones in kidney are scanty. In a prospective study, we aimed to determine the expression of Haemagglutinins, (Type 1 and P fimbriae), Biofilm production and resistance pattern to common antibiotics of Uropathogenic E.coli (UPEC) isolates from Community acquired Acute Urinary Tract Infection(CAUTI) and Urolithiasis.
Materials and Methods:
A total of 43 UPEC isolates, 23 mid-stream urine (MSU) samples from patients with CAUTI attending Out Patient Departments and 20 from renal calculi of urolithiasis patients at the time of Percutaneous nephrolithostomy (PCNL) were included in the study and the expression of Haemagglutinins,(Type 1 and P fimbriae), Biofilm production and resistance pattern to common antibiotics was assessed.
A total of 43 UPEC isolates 23 from CAUTI and 20 from renal calculi were tested for production of biofilm and hemagglutinins. In CAUTI, biofilm producers were 56.52% and hemagglutinins were detected in all isolates 100%. In urolithiasis, biofilm producers were 100% but hemagglutinins were detected only in 70% of isolates. All isolates were resistant to multiple antibiotics used. CAUTI isolates were susceptible to 3rd generation cephalosporins, whereas urolithiasis isolates were resistant to 3rd generation cephalosporins and 25% were Extended Spectrum Beta Lactamases ESBL producers.
HA mediated by type 1 fimbriae plays an important role in CAUTI (P < 0.001 highly significant), whereas, in chronic conditions like urolithiasis, biofilm plays an important role in persistence of infection and the role of hemagglutinins is less.
PMCID: PMC3822341  PMID: 24235787
Biofilm; hemagglutination; mannose-resistant hemagglutination; mannose-sensitive hemagglutination; urolithiasis; uropathogenic Escherichia coli
2.  Multidrug-Resistance and Extended Spectrum Beta-Lactamase Production in Uropathogenic E. Coli which were Isolated from Hospitalized Patients in Kolkata, India 
Background and Objective: Urinary Tract Infections (UTIs) are mostly caused by Escherichia coli. The appropriate therapy demands a current knowledge on the antimicrobial susceptibility pattern amongst these pathogens, as an inappropriate use of antibiotics may lead to complications and treatment failure. The UTIs which are caused by multidrug resistant Extended-Spectrum Beta-Lactamase (ESBL) producing bacteria further pose a severe problem, as the treatment options are limited. The aim of this study was to identify the pattern of multi drug resistance amongst the uropathogenic E. coli (UPEC) isolates which were obtained from hospitalized patients.
Materials and Methods: Forty UPEC were isolated from 200 urine samples of hospitalized patients who were clinically suspected for UTIs. Antimicrobial susceptibility screening was performed by using 16 antibiotics, by the Kirby Bauer disk diffusion technique. The isolates which were resistant to the third generation cephalosporins were subjected to the ESBL confirmatory test by using drug and drug-inhibitor combination disks by following the CLSI guidelines.
Results: All the 40 isolates except three were multidrug resistant. They showed the highest sensitivities for nitrofurantoin (72.5%) and amikacin (70%). A high level of resistance was observed against ampicillin (97.5%), nalidixic acid and cefelexin (95%), amoxicillin (92.5%), cotrimoxazole (82.5%) and ciprofloxacin (80%) respectively. Thirty different antibiotic resistance patterns were observed against the different antibiotics. Twenty-eight out of the 40 isolates were resistant to the third generation cephalosporins. However, the phenotypic test for the ESBL confirmation indicated that eighteen out of the twenty-eight isolates were ESBL producers and that eleven different drug resistance patterns were observed amongst them.
Conclusions: Therefore, this study accounts for the varied multidrug resistance pattern amongst the uropathogenic E. coli which were isolated from hospitalized patients in Kolkata, an eastern region of India. Nitrofurantoin and amikacin should be assigned as potent drugs to treat this infection in this region of the country. These varied resistance patterns present major therapeutic and infection control challenges and they suggest a heterogeneous population of the uropathogenic E. coli isolates which circulate in this sector of India.
PMCID: PMC3616554  PMID: 23634394
Antibiotic susceptibility; Urinary tract infection; Uropathogenic E. coli; Extended- spectrum beta lactamase
3.  The Isolation and the Biofilm Formation of Uropathogens in the Patients with Catheter Associated Urinary Tract Infections (UTIs) 
Urinary tract infections are the most commonly acquired bacterial infections and they account for an estimated 25-40% of the nosocomial infections. The microbial biofilms pose a public health problem for the persons who require indwelling medical devices, as the microorganisms in the biofilms are difficult to treat with antimicrobial agents.
The present study included the isolation and the biofilm formation of the uropathogens in patients with catheter associated urinary tract infections.
Methods and Materials
This prospective analysis which was carried out over a period of two months, included 50 urine samples from catheterized patients with symptoms of UTI. Following their isolation and identification, all the isolates were subjected to the biofilm detection by the tube adherence method and the Congo Red agar method.
E.coli was found to be the most frequently isolated uropathogen 35(70%), followed by Klebsiella pneumoniae 8(16%), Pseudomona aeruginosa 2(4%), Acinetobacter spp 1(2%), coagulase negative Staphylococci 3(6%) and Enterococci spp 1(2%). In the current study, 30 (60%) strains were positive in vitro for the biofilm production.
To conclude, there was significant bacteriuria in all the symptomatic catheterized patients and E.coli was the most frequent isolate. Diabetes (44%) was the most common factor which was associated with the UTIs in the catheterized patients.
PMCID: PMC3527774  PMID: 23285434
Biofilm; Uropathogens; The tube adherence method; The Congo Red agar method
4.  Antibiograms from community-acquired uropathogens in Gulu, northern Uganda - a cross-sectional study 
BMC Infectious Diseases  2013;13:193.
Urinary tract infections (UTI) are common in clinical practice and empirical treatment is largely employed due to predictability of pathogens. However, variations in antibiotic sensitivity patterns do occur, and documentation is needed to inform local empirical therapy. The current edition of the Uganda Clinical Guidelines recommends amoxicillin or cotrimoxazole as choice drugs for empirical treatment of community-acquired UTI. From our clinical observations, we suspected that this recommendation was not effective in our setting. In order to examine validity, we sought to identify bacteria from community-acquired infections and determine their susceptibility against these antibiotics plus a range of potentially useful alternatives for treatment of UTI.
A cross-sectional study of mid-stream urine collected from 339 symptomatic patients over a three-month period at Gulu regional referral hospital. Qualitative culture and identification of bacteria and antibiotic sensitivity testing using the modified Kirby-Bauer disk diffusion method was done. Participants’ demographic and clinical characteristics were collected using a standard form. Results were analyzed by simple proportions among related variables and confidence intervals computed using binomial exact distribution.
Eighty two cultures were positive for UTI. Staphylococcus spp (46.3%) and Escherichia coli (39%) were the most common pathogens. There was high resistance to cotrimoxazole (73.2%), nalidixic acid (52.4%) and amoxicillin (51.2%). The most favorable antibiograms were obtained with gentamicin, amoxicillin-clavulanate and levofloxacin where 85.4%, 72.0%, 67.1% of isolates respectively, were either sensitive or intermediate. Only 51% of isolates were sensitive to ciprofloxacin.
There was high resistance to most antibiotics tested in this study. The recommendations contained in the current edition of the Uganda Clinical Guidelines are not in tandem with antibiotic sensitivity pattern of uropathogens seen in our setting. Amoxicillin-clavulanate or gentamicin should be considered for replacement of amoxicillin and cotrimoxazole for empirical treatment of UTI in our setting.
PMCID: PMC3643887  PMID: 23627344
Antibiograms; Antibiotic resistance; Uropathogens; Empirical treatment; Uganda
5.  The influence of diabetes mellitus on the spectrum of uropathogens and the antimicrobial resistance in elderly adult patients with urinary tract infection 
The role of Diabetes mellitus (DM) in the etiology and in the antimicrobial resistance of uropathogens in patients with urinary tract infection has not been well clarified. For this reason we have evaluated the spectrum of uropathogens and the profile of antibiotic resistance in both diabetic and non diabetic patients with asymptomatic urinary tract infection (UTI).
Urinary isolates and their patterns of susceptibility to the antimicrobials were evaluated in 346 diabetics (229 females and 117 males) and 975 non diabetics (679 females and 296 males) who were screened for significant bacteriuria (≥105 CFU/mL urine). The mean age of diabetic and non diabetic patients was respectively 73.7 yrs ± 15 S.D. and 72.7 ± 24 (p = NS).
Most of our patients had asymptomatic UTI. The most frequent causative organisms of bacteriuria in females with and without DM were respectively : E. coli 54.1% vs 58.2% (p = NS), Enterococcus spp 8.3% vs 6.5% (p = NS), Pseudomonas spp 3.9 vs 4.7% (p = NS). The most frequent organisms in diabetic and non diabetic males were respectively E. coli 32.5% vs 31.4% (p = NS), Enterococcus spp 9.4% vs 14.5% (p = NS), Pseudomonas spp 8.5% vs 17.2% (p = <0.02). A similar isolation rate of E. coli, Enterococcus spp and Pseudomonas spp was also observed in patients with indwelling bladder catheter with and without DM. No significant differences in resistance rates to ampicillin, nitrofurantoin, cotrimoxazole and ciprofloxacin of E. coli and Enteroccus spp were observed between diabetic and non diabetic patients.
In our series of patients with asymptomatic UTI (mostly hospital acquired), diabetes mellitus per se does not seem to influence the isolation rate of different uropathogens and their susceptibility patterns to antimicrobials.
PMCID: PMC1434753  PMID: 16545130
6.  Retrospective analysis of antibiotic resistance pattern to urinary pathogens in a Tertiary Care Hospital in South India 
The distribution of uropathogens and their susceptibility pattern to antibiotics vary regionally and even in the same region, they change over time. Therefore, the knowledge on the frequency of the causative microorganisms and their susceptibility to various antibiotics are necessary for a better therapeutic outcome.
The aim was to study the frequency and distribution of uropathogens and their resistance pattern to antibiotics in a tertiary care hospital.
Settings and Design:
Retrospective study for a period of 1 year from January 2011 to December 2011 in a tertiary care hospital.
Materials and Methods:
The culture and sensitivity data of the uropathogens from suspected cases of UTI were collected from the records of Microbiology Department for study period. Midstream urine samples were processed for microscopy and culture, and the organisms were identified by standard methods. Antibiotic susceptibility was carried out by Kirby-Bauer disk diffusion method according to Clinical and Laboratory Standards Institute guidelines. Descriptive statistics were used to analyze the data.
Of 896 urine samples, 348 (38.84%) samples were positive for urine culture. Escherichia coli (52.59%) was the most common organism followed by Klebsiella. E. coli was least resistant to imipenem (8%) and amikacin (16%) and was highly resistant to co-trimoxazole (69%) and ampicillin (86%). Klebsiella species were least resistant to amikacin (26%) and were highly resistant to ampicillin (92%). The overall resistance pattern of antibiotics to uropathogens was the highest to nalidixic acid (79%) followed by co-trimoxazole (75%) and ampicillin (72%). Good susceptibility was seen with imipenem and cephalosporins.
E. coli is still the most common uropathogen. Nalidixic acid, ampicillin, co-trimoxazole, and first-generation fluoroquinolones have limited value for the treatment of UTI. Sensitivity to imipenem and amikacin are still retained and may be prescribed for complicated UTI. Routine monitoring of drug resistance pattern will help to identify the resistance trends regionally. This will help in the empirical treatment of UTIs to the clinicians.
PMCID: PMC4194940  PMID: 25316990
Antibiotics; resistance; uropathogens
7.  Etiology and antibiotic resistance patterns of community-acquired urinary tract infections in J N M C Hospital Aligarh, India 
Urinary tract infections (UTIs) remain the common infections diagnosed in outpatients as well as hospitalized patients. Current knowledge on antimicrobial susceptibility pattern is essential for appropriate therapy. Extended-Spectrum beta-Lactamase (ESBL) producing bacteria may not be detected by routine disk diffusion susceptibility test, leading to inappropriate use of antibiotics and treatment failure. The aim of this study was to determine the distribution and antibiotic susceptibility patterns of bacterial strains isolated from patients with community acquired urinary tract infections (UTIs) at Aligarh hospital in India as well as identification of ESBL producers in the population of different uropathogens.
Urinary isolates from symptomatic UTI cases attending to the JN Medical College and hospital at Aligarh were identified by conventional methods. Antimicrobial susceptibility testing was performed by Kirby Bauer's disc diffusion method. Isolates resistant to third generation cephalosporin were tested for ESBL production by double disk synergy test method.
Of the 920 tested sample 100 samples showed growth of pathogens among which the most prevalent were E. coli (61%) followed by Klebsiella spp (22%). The majority (66.66%) of the isolates were from female while the remaining were from male. Among the gram-negative enteric bacilli high prevalence of resistance was observed against ampicillin and co-trimoxazole. Most of the isolates were resistant to 4 or more number of antibiotics. Forty two percent of isolates were detected to produce ESBL among which 34.42 % were E. coli isolates.
This study revealed that E. coli was the predominant bacterial pathogen of community acquired UTIs in Aligarh, India. It also demonstrated an increasing resistance to Co-trimoxazole and production of extended spectrum β-lactamase among UTI pathogens in the community. This study is useful for clinician in order to improve the empiric treatment.
PMCID: PMC1852324  PMID: 17378940
8.  Clinical profile of urinary tract infections in diabetics and non-diabetics 
The risk of urinary tract infection (UTI) is higher in diabetics compared to non-diabetics. The aetiology and the antibiotic resistance of uropathogens have been changing over the past years. Hence the study was undertaken to determine if there are differences in clinical and microbiological features of UTI between diabetic and non-diabetic subjects, to study the influence of diabetes mellitus on the uropathogens and antibiotic sensitivity pattern in patients with UTI.
A total of 181 diabetics (83 males and 98 females) and 124 non-diabetic subjects (52 males and 72 females) with culture positive UTI were studied. Patients with negative urine culture (n= 64), those diagnosed and treated outside (n= 83) and not willing to participate in the study (n= 24) were excluded.
Almost 30 per cent of the patients (both diabetics and nondiabetics) presented with asymptomatic bacteriuria and the prevalence of pyelonephritis was significantly higher (p= 0.04) in diabetics compared to non-diabetic patients. The majority of the diabetics with UTI (87.14 per cent) had glycosylated haemoglobin (HbA1c) > 6.5 per cent with p < 0.001. The isolation rate of Escherichia coli (E. coli) from urine culture was higher (64.6 per cent) among diabetic patients followed by Klebsiella (12.1 per cent) and Enterococcus (9.9 per cent). The prevalence of extendedspectrum beta-lactamase (ESBL) producing E.coli was significantly higher in diabetics (p= 0.001) compared to nondiabetics. E.coli showed maximum sensitivity to carbapenems in both diabetic and non-diabetic subjects and least susceptibility to ampicillin.
The prevalence of pyelonephritis is significantly higher in diabetics than in non-diabetic subjects, with E. coli being the most common isolate. Elevated glycosylated hemoglobin (HbA1c) predisposes diabetics to UTI. Investigation of bacteriuria in diabetic patients for urinary tract infection is important for treatment and prevention of renal complications.
PMCID: PMC3920469  PMID: 24567764
Urinary tract infection; Asymptomatic bacteriuria; Uropathogens; Diabetic patients; E. coli
9.  Distribution and Antimicrobial Susceptibility Pattern of Bacterial Pathogens Causing Urinary Tract Infection in Urban Community of Meerut City, India 
ISRN Microbiology  2013;2013:749629.
Urinary tract infection is one of the common infections in the Indian community. Distribution and susceptibility of UTI-causing pathogens change according to time and place. This study was conducted to determine the distribution and antimicrobial susceptibility of uropathogens in the Indian community as well as to determine the effect of gender and age on the etiology of bacterial uropathogens. Clean catch midstream urine samples were collected from 288 patients of the age ranging from 15 to ≥48 years. Antimicrobial susceptibility was performed on all isolated bacteria by Kirby Bauer's disc diffusion method. The multiple antibiotic resistance (MAR) index of each antibiotic was calculated. The UTI prevalence was 53.82% in patients; however, the prevalence was significantly higher in females than in males (females: 73.57%; males: 35.14%; P = 0.000). Females within the age group of 26–36 years and elderly males of ≥48 years showed higher prevalence of UTI. Gram negative bacteria (90.32%) were found in high prevalence than Gram positive (9.68%). Escherichia coli (42.58%) was the most prevalent gram negative isolate. Nitrofurantoin (78.71%) was found the most resistant drug among all uropathogens. Tested carbapenems were found the most susceptible drug against isolated uropathogens which showed 92.26% and 84.52% susceptibility, respectively.
PMCID: PMC3830820  PMID: 24288649
10.  Antibiotic resistance pattern among common bacterial uropathogens with a special reference to ciprofloxacin resistant Escherichia coli 
Background & objectives:
The resistance of bacteria causing urinary tract infection (UTI) to commonly prescribed antibiotics is increasing both in developing as well as in developed countries. Resistance has emerged even to more potent antimicrobial agents. The present study was undertaken to report the current antibiotic resistance pattern among common bacterial uropathogens isolated in a tertiary care hospital in south India, with a special reference to ciprofloxacin.
A total of 19,050 consecutive urine samples were cultured and pathogens isolated were identified by standard methods. Antibiotic susceptibility was done by Kirby Bauer disk diffusion method. The clinical and demographic profile of the patients was noted.
Of the 19,050 samples, 62 per cent were sterile, 26.01 per cent showed significant growth, 2.3 per cent showed insignificant growth and 9.6 per cent were found contaminated. Significant association (P<0.001) of prior use of antibiotics in males, UTI in adults, gynaecological surgery in females, obstructive uropathy in males and complicated UTI in females with the occurrence of UTI with ciprofloxacin resistant Escherichia coli was noted. Significant association was noted in females with prior antibiotics, with prior urological surgery and in males with prior complicated UTI. There was no significant association with diabetes mellitus with the occurrence of UTI with ciprofloxacin resistant E. coli. Fluoroquinolone resistance was found to increase with age.
Interpretations & conclusions:
Ciprofloxacin resistance has emerged due to its frequent use. This resistance was seen more in the in-patients, elderly males and females. Also the resistance to other antibiotics was also high. Increasing antibiotic resistance trends indicate that it is imperative to rationalize the use of antimicrobials in the community and also use these conservatively.
PMCID: PMC3573607  PMID: 23287133
Ciprofloxacin; Escherichia coli; minimum inhibitory concentration; urinary tract infection
11.  Antimicrobial resistance in urinary isolates from inpatients and outpatients at a tertiary care hospital in South-Kivu Province (Democratic Republic of Congo) 
BMC Research Notes  2014;7:374.
The rate of antimicrobial resistant isolates among pathogens causing urinary tract infections (UTIs) in Democratic Republic of Congo (DRC) is not known. The aim of the current study was to determine this rate at the Bukavu Provincial General Hospital (province of South-Kivu, DRC).
A total of 643 isolates (both from inpatients and outpatients) collected from September 2012 to August 2013 were identified using biochemical methods, and tested for antimicrobial susceptibility. The isolates were further screened for Extended-Spectrum Beta-Lactamases (ESBL) production. Beta-lactamase AmpC phenotype was investigated in 20 antibiotic-resistant isolates.
Escherichia coli (58.5%), Klebsiella spp. (21.9%) and Enterobacter spp. (16.2%) were the most frequent uropathogens encountered. Rare uropathogens included Citrobacter spp., Proteus spp., and Acinetobacter spp. Resistance was significantly more present in inpatients isolates (22.1% of isolates) when compared to outpatients isolates (8.4% of isolates), (p-value <0.001). Antibiotic-resistant isolates displayed resistance to common antimicrobial drugs used for UTIs treatment in South Kivu province, namely: ciprofloxacin, ampicillin and third generation cephalosporins. ESBL-phenotype was present in 92.9% of antibiotic-resistant isolates. Only amikacin, nitrofurantoin and imipenem displayed satisfactory activity against antibiotic resistant isolates.
This study confirms the presence of antibiotic-resistant uropathogens (mainly ESBL-producers isolates) at the Bukavu General Hospital. This study should serve as a wake-up call and help to raise awareness about the threat to public health of antibiotic resistance in this DRC province.
PMCID: PMC4074390  PMID: 24943866
Urinary tract infections; South-Kivu; Democratic Republic of Congo; Multidrug resistance; Prevalence; Extended spectrum beta-lactamases; Antibotics
12.  Evaluating antibiotics for use in medicine using a poloxamer biofilm model 
Wound infections, due to biofilms, are a constant problem because of their recalcitrant nature towards antibiotics. Appropriate antibiotic selection for the treatment of these biofilm infections is important. The traditional in vitro disc diffusion method for antibiotic selection uses bacterial cultures grown on agar plates. However, the form of bacterial growth on agar is not representative of how bacteria grow in wounds and other tissue sites as here bacteria grow naturally in a biofilm. The aim of this research was to test a more appropriate method for testing antimicrobial efficacy on biofilms and compare with the standard methods used for antibiotic sensitivity testing.
Outer Membrane Protein analysis was performed on E.coli, Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis and Acinetobacter juni when grown on Mueller Hinton agar ('quasi-biofilm state') and 30% Poloxamer hydrogel ('true- biofilm state). Susceptibility to antibiotics on 28 clinical isolates was determined using the modified Kirby Bauer disc diffusion method, on agar and 30% Poloxamer.
Similar outer membrane proteins [OMPs] were identified in bacteria grown in a biofilm state and on a 30% poloxamer hydrogel, which were very different to the OMPs identified in bacteria grown on Mueller-Hinton agar and broth. There was a significant difference between the means of the clearance zones around the antibiotic discs on standard agar and poloxamer gels [P < 0.05]. The zones of clearance were generally smaller for poloxamer-grown bacteria than those grown on standard agar. Diffusion distances of various antibiotics through agar and 30% poloxamer showed no significant difference [P > 0.05].
The findings of this experiment suggest that poloxamer gel could be used as an appropriate medium on which to conduct biofilm antibiotic susceptibility tests as it enables bacteria to be grown in a state representative of the infected surface from which the culture was taken.
PMCID: PMC1804279  PMID: 17302978
13.  Biofilm and fluoroquinolone resistance of canine Escherichia coli uropathogenic isolates 
BMC Research Notes  2014;7:499.
Escherichia coli is the most common uropathogen involved in urinary tract infection (UTI). Virulence of strains may differ, and may be enhanced by antimicrobial resistance and biofilm formation, resulting in increased morbidity and recurrent infections. The aim of this study was to evaluate the in vitro biofilm forming capacity of E. coli isolates from dogs with UTI, by using fluorescent in situ hybridization, and its association with virulence genes and antimicrobial resistance.
The proportion of biofilm-producing isolates significantly increased with the length of incubation time (P < 0.05). Biofilm production was significantly associated with fluoroquinolone resistance at all incubation time points and was independent of the media used (P < 0.05). Biofilm production was not associated with cnf1, hly, pap and sfa genes (P > 0.05), but was significantly associated with afa, aer and the β-lactamase genes (P < 0.05).
To the best of our knowledge, this is the first report showing significant association between biofilm production and fluoroquinolone resistance in E. coli isolates from dogs with UTI. Biofilm formation may contribute to UTI treatment failure in dogs, through the development of bacterial reservoirs inside bladder cells, allowing them to overcome host immune defenses and to establish recurrent infections.
PMCID: PMC4132243  PMID: 25099929
Biofilm; Dogs; Escherichia coli; Fluoroquinolone resistance; Urinary tract infection
14.  Role of the rapA Gene in Controlling Antibiotic Resistance of Escherichia coli Biofilms▿ †  
Antimicrobial Agents and Chemotherapy  2007;51(10):3650-3658.
By using a high-throughput screening method, a mutant of a uropathogenic Escherichia coli strain affected in the rapA gene was isolated. The mutant formed normal-architecture biofilms but showed decreased penicillin G resistance, although the mutation did not affect planktonic cell resistance. Transcriptome analysis showed that 22 genes were down-regulated in the mutant biofilm. One of these genes was yhcQ, which encodes a putative multidrug resistance pump. Mutants with mutations in this gene also formed biofilms with decreased resistance, although the effect was less pronounced than that of the rapA mutation. Thus, an additional mechanism(s) controlled by a rapA-regulated gene(s) was involved in wild-type biofilm resistance. The search for this mechanism was guided by the fact that another down-regulated gene in rapA biofilms, yeeZ, is suspected to be involved in extra cell wall-related functions. A comparison of the biofilm matrix of the wild-type and rapA strains revealed decreased polysaccharide quantities and coverage in the mutant biofilms. Furthermore, the (fluorescent) functional penicillin G homologue Bocillin FL penetrated the mutant biofilms more readily. The results strongly suggest a dual mechanism for the wild-type biofilm penicillin G resistance, retarded penetration, and effective efflux. The results of studies with an E. coli K-12 strain pointed to the same conclusion. Since efflux and penetration can be general resistance mechanisms, tests were conducted with other antibiotics. The rapA biofilm was also more sensitive to norfloxacin, chloramphenicol, and gentamicin.
PMCID: PMC2043260  PMID: 17664315
15.  Resistance profile for pathogens causing urinary tract infection in a pediatric population, and antibiotic treatment response at a University Hospital, 2010-2011  
Colombia Médica : CM  null;45(1):39-44.
Urinary tract infection (UTI) is one of the most common bacterial infections in childhood and causes acute and chronic morbidity and long-term hypertension and chronic kidney disease.
To describe the demographic characteristics, infectious agents, patterns of antibiotic resistance, etiologic agent and profile of susceptibility and response to empirical treatment of UTI in a pediatric population.
This is a descriptive, retrospective study.
Included in the study were 144 patients, 1:2.06 male to female ratio. The most common symptom was fever (79.9%) and 31.3% had a history of previous UTI. 72.0% of the patients had positive urine leukocyte count (>5 per field), urine gram was positive in 85.0% of samples and gram negative bacilli accounted for 77.8% for the total pathogens isolated. The most frequent uropathogens isolated were Escherichia coli and Klebsiella pneumoniae. Our E.coli isolates had a susceptibility rate higher than 90% to most of the antibiotics used, but a resistance rate of 42.6% to TMP SMX and 45.5% to ampicillin sulbactam. 6.3% of E. coli was extended-spectrum beta-lactamases producer strains. The most frequent empirical antibiotic used was amikacin, which was used in 66.0% of the patients. 17 of 90 patients who underwent voiding cistouretrography (VCUG) had vesicoureteral reflux.
This study revealed that E. coli was the most frequent pathogen of community acquired UTI. We found that E. coli and other uropathogens had a high resistance rate against TMP SMX and ampicillin sulbactam. In order to ensure a successful empirical treatment, protocols should be based on local epidemiology and susceptibility rates.
PMCID: PMC4045227  PMID: 24970958
Urinary tract infection; drug resistance; bacterial; Escherichia coli
16.  Prevalence of extended spectrum beta lactamase producing Escherichia coli and Klebsiella pneumoniae urinary isolates in a tertiary care hospital in Kathmandu, Nepal 
BMC Research Notes  2013;6:487.
Escherichia coli and Klebsiella pneumoniae are the major bacterial pathogens being isolated and reported from mid stream urine (MSU) specimens, globally. These uropathogens are mostly implicated as the major extended spectrum beta-lactamase (ESBL) producers, severely limiting the therapeutic management in cases of urinary tract infections. Limited studies had been reported from Nepal investigating the ESBL producers among uropathogens. This study was designed to assess the prevalence of ESBL producing E.coli and K. pneumoniae in urinary isolates at a centrally located major tertiary care hospital in Kathmandu valley, Nepal.
Between September 2011 and May 2012, during the nine months period, 6308 MSU specimens were collected aseptically from the same number of clinically suspected patients of urinary tract infections. The samples were cultured on MacConkey agar and blood agar. The isolates with significant bacteriuria (105 CFU / ml) were identified based on standard laboratory procedures. Antimicrobial susceptibility tests were carried out using various antimicrobial discs alongwith ceftriaxone on E.coli and K. pneumoniae isolates by Kirby Bauer disc diffusion method as per the recommendations of CLSI. On initial screening with ceftriaxone (30 μg) disc showing resistance was then confirmed for ESBL production by phenotypic confirmatory disc diffusion test (PCDDT) using ceftazidime (30 ug) and ceftazidime + clavulanic acid (30 μg + 10ug) disc as per guidelines of CLSI (2011).
Out of a total of 6308 MSU specimens investigated for significant bacteriuria, E.coli isolates were 444 (7.04%) and K.pneuminiae were 145 (2.3%) making a total of 589 (9.34%). Initial screening with ceftriaxone disc revealed 155 isolates of E.coli and 70 isolates of K.pneumoniae to be resistant. Further testing by PCDDT method showed 60/444 (=13.51%) of E. coli and 24/145 (=16.55%) of K. pneumoniae isolates to be confirmed ESBL producers. These ESBL – producer uropathogens showed high degree of resistance to ceftriaxone (100.0%), amoxycillin, fluoroquinolones and co-trimoxazole.
An emerging and moderately high prevalence of ESBL-producing E. coli and K. pneumoniae was observed and confirmed in the urinary isolates investigated. It is essential to have a regular and routine monitoring of ESBL producing clinical isolates in laboratory practice.
PMCID: PMC4222089  PMID: 24274894
Antimicrobial resistance; Extended spectrum beta lactamase (ESBL); Escherichia coli; Klebsiella pneumoniae; Mid stream urine (MSU); Prevalence; Urinary isolates
17.  Urinary Tract Infection: Aetiology and Antimicrobial Resistance Pattern in Infants From A Tertiary Care Hospital in Northern India 
Introduction: Urinary tract infection (UTI) is one of the most common bacterial infections in childhood. Present study was undertaken to determine the occurrence of the uropathogens and their antimicrobial susceptibility pattern in infants (< 1yr) suspected with UTI.
Materials and Methods: This study was conducted in the Microbiology Department on urine samples received from infants for a period of two years from September 2011 to August 2013.
Results: Culture positivity rate was found to be 15.7%. There was an overall male preponderance in cases of UTI (70.1%). Most common bacterial isolate was E.coli (45.4%) followed by Klebsiella (16.7%) and Enterococcus spp (13.2%). Isolation of candida was 21.1%, maximum from ICU (63.1%). Maximum gram negative isolates (50%) showed high resistance to gentamicin, amikacin, cefotaxime and norfloxacin while most of the isolates (5%) were found susceptible to nitrofurantoin and piperacillin-tazobactam. 45.1% of gram negative bacilli were ESBL producer. We recommend continuous monitoring of changes in bacterial pathogens causing UTI and antibiotic sensitivity in each area for effective treatment of UTI.
Conclusion: Since antimicrobial resistance is a major problem, such study will help in formulating a strict antibiotics prescription policy in our country.
PMCID: PMC4253157  PMID: 25478339
Antibiotic sensitivity; Antibiotic prescription policy; Infant UTI
18.  A Study on Device-Related Infections with Special Reference to Biofilm Production and Antibiotic Resistance 
Indwelling medical devices (IMDs) in critical patients are vulnerable to colonization by biofilm producing bacteria. Complex characteristics of bacterial biofilms promote antibiotic resistance, leading to the emergence of resistant device-related infections (DRI), which pose new challenges in their management.
Materials and Methods:
The study was done on 135 hospitalized (Intensive care units) pediatric patients with IMDs (intravascular catheter, urinary catheter, and endotracheal tube) to determine the device-specific infection rates. Biofilm formations were demonstrated by the tube method and by scanning electron microscopy (SEM). Bacteria in biofilms were identified by the standard conventional methods and tested for antibiotic resistance. We also detected the presence of extended spectrum β-lactamases (ESβLs), particularly, blaCTX-M, in gram-negative isolates.
The rates of biofilm-based catheter-related blood stream infections (CRBSI), catheter-associated urinary tract infections (CAUTI), and Ventilator Associated Pneumonia (VAP), in our study, were 10.4, 26.6, and 20%. Biofilm formation by the tube method correlated well with the SEM findings. A majority of infections were caused by Klebsiella pneumoniae followed by Staphylococcal biofilms. A high percentage (85.7%, 95% confidence interval 64.5 to 95.8%) of biofilm producing bacterial isolates, causing infection, were multidrug resistant. Many biofilm producing gram-negative isolates were ESβLs producers, and a majority particularly harbored blaCTX-M, among the ESβLs genotypes.
The incidence of resistant device-related infections, predominantly caused by biofilm producing bacteria, is rising. The tube method is an effective screening method to test biofilm production, where sophisticated microscopy facilities are not available. The varying resistance pattern of organisms isolated in our setup, emphasizes the importance of studying the pattern of infection in every setting and providing antibiotic guidelines in the management of such infections.
PMCID: PMC3543538  PMID: 23326076
Biofilms; Device-related infection; ESβL; MRSA; Scanning electron microscopy
19.  Biofilm formation by clinical isolates and the implications in chronic infections 
Biofilm formation is a major virulence factor contributing to the chronicity of infections. To date few studies have evaluated biofilm formation in infecting isolates of patients including both Gram-positive and Gram-negative multidrug-resistant (MDR) species in the context of numerous types of infectious syndromes. Herein, we investigated the biofilm forming capacity in a large collection of single patient infecting isolates and compared the relationship between biofilm formation to various strain characteristics.
The biofilm-forming capacity of 205 randomly sampled clinical isolates from patients, collected from various anatomical sites, admitted for treatment at Brooke Army Medical Center (BAMC) from 2004–2011, including methicillin-resistant/methicillin susceptible Staphylococcus aureus (MRSA/MSSA) (n=23), Acinetobacter baumannii (n=53), Pseudomonas aeruginosa (n=36), Klebsiella pneumoniae (n=54), and Escherichia coli (n=39), were evaluated for biofilm formation using the high-throughput microtiter plate assay and scanning electron microscopy (SEM). Relationships between biofilm formation to clonal type, site of isolate collection, and MDR phenotype were evaluated. Furthermore, in patients with relapsing infections, serial strains were assessed for their ability to form biofilms in vitro.
Of the 205 clinical isolates tested, 126 strains (61.4%) were observed to form biofilms in vitro at levels greater than or equal to the Staphylococcus epidermidis, positive biofilm producing strain, with P. aeruginosa and S. aureus having the greatest number of biofilm producing strains. Biofilm formation was significantly associated with specific clonal types, the site of isolate collection, and strains positive for biofilm formation were more frequently observed to be MDR. In patients with relapsing infections, the majority of serial isolates recovered from these individuals were observed to be strong biofilm producers in vitro.
This study is the first to evaluate biofilm formation in a large collection of infecting clinical isolates representing diverse types of infections. Our results demonstrate: (1) biofilm formation is a heterogeneous property amongst clinical strains which is associated with certain clonal types, (2) biofilm forming strains are more frequently isolated from non-fluid tissues, in particular bone and soft tissues, (3) MDR pathogens are more often biofilm formers, and (4) strains from patients with persistent infections are positive for biofilm formation.
PMCID: PMC3568419  PMID: 23356488
Biofilm formation; Clinical isolates; Chronic infection; Multidrug-resistant; MRSA
20.  Community Behavior and Amyloid-associated Phenotypes among a Panel of Uropathogenic E. coli 
Uropathogenic E. coli (UPEC) are the major causative agents of urinary tract infection and engage in a coordinated genetic and molecular cascade to colonize the urinary tract. Disrupting the assembly and/or function of virulence factors and bacterial biofilms has emerged as an attractive target for the development of new therapeutic strategies to prevent and treat urinary tract infection, particularly in the era of increasing antibiotic resistance among human pathogens. UPEC vary widely in their genetic and molecular phenotypes and more data are needed to understand the features that distinguish isolates as more or less virulent and as more robust biofilm formers or poor biofilm formers. Curli are extracellular functional amyloid fibers produced by E. coli that contribute to pathogenesis and influence the host response during urinary tract infection (UTI). We have examined the production of curli and curli-associated phenotypes including biofilm formation among a specific panel of human clinical UPEC that has been studied extensively in the mouse model of UTI. Motility, curli production, and curli-associated biofilm formation attached to plastic were the most prevalent behaviors, shared by most clinical isolates. We discuss these results in the context on the previously reported behavior and phenotypes of these isolates in the murine cystitis model in vivo.
PMCID: PMC3932320  PMID: 24239885
curli; functional amyloid; biofilm; amyloid; Congo red; uropathogenic E. coli
21.  Antimicrobial resistance in pathogens causing urinary tract infections in a rural community of Odisha, India 
Antimicrobial resistance of urinary tract pathogens has increased worldwide. Empiric treatment of community-acquired urinary tract infection (CA-UTI) is determined by antimicrobial resistance patterns of uropathogens in a population of specific geographical location.
This study was conducted to determine the prevalence of CA-UTI in rural Odisha, India, and the effect of gender and age on its prevalence as well as etiologic agents and the resistance profile of the bacterial isolates.
Materials and Methods:
Consecutive clean-catch mid-stream urine samples were collected from 1670 adult patients. The urine samples were processed and microbial isolates were identified by conventional methods. Antimicrobial susceptibility testing was performed on all bacterial isolates by Kirby Bauer's disc diffusion method.
The prevalence of UTI was significantly higher in females compared with males (females 45.2%, males 18.4%, OR = 2.041, 95% CI = 1.64-2.52, P ≤ 0.0001). Young females within the age group of 18-37 years and elderly males (≥68 years) showed high prevalence of UTI. Escherichia coli (68.8%) was the most prevalent isolate followed by Enterococcus spp. (9.7%). Amikacin and nitrofurantoin were the most active antimicrobial agents which showed low resistance rate of 5.8% and 9.8%, respectively.
Our study revealed E. coli as the pre-dominant bacterial pathogen. Nitrofurantoin should be used as empirical therapy for uncomplicated CA-UTIs. In the Indian setting, routine urine cultures may be advisable, since treatment failure is likely to occur with commonly used antimicrobials. Therefore, development of regional surveillance programs is necessary for implementation of national CA-UTI guidelines.
PMCID: PMC3663160  PMID: 23723727
Antimicrobial; community-acquired urinary tract infection; Escherichia coli; prevalence; resistance
22.  Multiple Species of Trichosporon Produce Biofilms Highly Resistant to Triazoles and Amphotericin B 
PLoS ONE  2014;9(10):e109553.
Invasive infections caused by Trichosporon spp. have increased considerably in recent years, especially in neutropenic and critically ill patients using catheters and antibiotics. The genus presents limited sensitivity to different antifungal agents, but triazoles are the first choice for treatment. Here, we investigated the biofilm production and antifungal susceptibility to triazoles and amphotericin B of 54 Trichosporon spp. isolates obtained from blood samples (19), urine (20) and superficial mycosis (15). All isolates and 7 reference strains were identified by sequence analysis and phylogenetic inferences of the IGS1 region of the rDNA. Biofilms were grown on 96-well plates and quantitation was performed using crystal violet staining, complemented with Scanning Electron Microscopy (SEM). Susceptibility tests for fluconazole, itraconazole, voriconazole and amphotericin B were processed using the microdilution broth method (CLSI) for planktonic cells and XTT reduction assay for biofilm-forming cells. Our results showed that T. asahii was the most frequent species identified (66.7%), followed by T. faecale (11.1%), T. asteroides (9.3%), T. inkin (7.4%), T. dermatis (3.7%) and one T. coremiiforme (1.8%). We identified 4 genotypes within T. asahii isolates (G1, G3, G4 and G5) and 2 genotypes within T. faecale (G1 and G3). All species exhibited high adhesion and biofilm formation capabilities, mainly T. inkin, T. asteroides and T. faecale. Microscopy images of high biofilm-producing isolates showed that T. asahii presented mainly hyphae and arthroconidia, whereas T. asteroides exhibited mainly short arthroconidia and few filaments. Voriconazole exhibited the best in vitro activity against all species tested. Biofilm-forming cells of isolates and reference strains were highly resistant to all antifungals tested. We concluded that levels of biofilm formation by Trichosporon spp. were similar or even greater than those described for the Candida genus. Biofilm-forming cells were at least 1,000 times more resistant to antifungals than planktonic cells, especially to voriconazole.
PMCID: PMC4215839  PMID: 25360765
23.  Global Gene Expression Profiling of Asymptomatic Bacteriuria Escherichia coli during Biofilm Growth in Human Urine▿  
Infection and Immunity  2006;75(2):966-976.
Urinary tract infection (UTI) is an important health problem worldwide, with many millions of cases each year, and Escherichia coli is the most common organism causing UTI in humans. Also, E. coli is responsible for most infections in patients with chronic indwelling bladder catheter. The two asymptomatic bacteriuria (ABU) E. coli strains 83972 and VR50 are significantly better biofilm formers in their natural growth medium, human urine, than the two uropathogenic E. coli isolates CFT073 and 536. We used DNA microarrays to monitor the expression profile during biofilm growth in urine of the two ABU strains 83972 and VR50. Significant differences in expression levels were seen between the biofilm expression profiles of the two strains with the corresponding planktonic expression profiles in morpholinepropanesulfonic acid minimal laboratory medium and human urine; 417 and 355 genes were up- and down-regulated, respectively, during biofilm growth in urine of 83972 and VR50. Many genes involved in transcription and stress were up-regulated in biofilm-grown cells. The role in biofilm formation of four of the up-regulated genes, i.e., yceP, yqgA, ygiD, and aaeX, was investigated by creating single-knockout mutant versions of 83972 and VR50; all mutants showed reduced biofilm formation in urine by 18 to 43% compared with the wild type (P < 0.05). Also, the expression profile of strain 83972 in the human urinary tract partially overlaps with the biofilm expression profile.
PMCID: PMC1828481  PMID: 17145952
24.  The Antibiotic Susceptibility Patterns of Uropathogenic Escherichia Coli, With Special Reference to the Fluoroquinolones 
Context: The emergence of drug resistance to trimethoprim-sulfamethoxazole, the penicillins, cephalosporins, and fluoroquinolones by Uropathogenic Escherichia coli (UPEC) has limited the options for selecting the appropriate antibiotic for the treatment of urinary tract infections.
Aims: The The E. coli isolates, which were obtained from the culture of urine samples,were studied for their antibiotic resistance patterns, with special reference to the antimicrobial activity of the fluoroquinolones and the production of the extended spectrum β-lactamases. (ESBL), Settings and Design: This was a hospital based, prospective study which was done for a period of eighteen months.
Material and Methods: This study was done by using the standard culture techniques for urine samples, the modified Kirby-Bauer disk diffusion method for the antibiotic susceptibility testing and the disk diffusion method to confirm the ESBL production by the clinical isolates of E. coli in urine. The sensitivity pattern was correlated with the clinical condition and the presence of the risk factors.
The statistical analysis which was used: The statistical analysis was done by using the proportions of sensitive, resistant and intermediates. Descriptive statistics like the total, mean and percentage were done by using the Statistical Package for the Social Sciences (SPSS), version 15.0.
Results: The hospital isolates showed high degrees of resistance to the penicillins, cephalosporins, nalidixic acid and the fluoroquinolones, with 59% of the isolates being ESBL producers.
Conclusions: The incidence of the multidrug resistant strains of Escherichia coli has been steadily increasing over the past few years. The knowledge on the resistance pattern of the bacterial strains in a geographical area will help in guiding the appropriate and the judicious use of antibiotics. Also, the formulation of an appropriate hospital antibiotic policy will go a long way in controlling these infections.
PMCID: PMC3708190  PMID: 23905095
ESBL; UPEC; Fluoroquinolones; Escherichia coli
25.  Increasing ciprofloxacin resistance of isolates from infected urines of a cross-section of patients in Karachi 
BMC Research Notes  2012;5:696.
The objective of the research was to evaluate the current effectiveness of Ciprofloxacin on the uropathogens prevalent in infected urines of a cross-section of patients in Karachi, Pakistan.
An observational study conducted in a private diagnostic laboratory and its branches in key areas of Karachi City from February 2010 to July 2011. A total of 2963 consecutive urine samples were cultured on chocolate agar, CLED medium and selective EMB agar. Growth of possible uropathogens was noted, and compared retrospectively with earlier lab data of suggestive urine cultures (n = 1997) recorded during January 2009 and December 2009. The isolates were identified using routine procedures and the API 20 system and evaluated for their sensitivity to ciprofloxacin by Kirby-Bauer disk diffusion method. Data was subjected to statistical analysis on SPSS version 16. Out of the present-day culture-positive urines, 2409 (80.4%) yielded gram-negative rods, and 554 (18.5%) gram-positive cocci. E.coli (43.1%) was most frequent, followed by Klebsiella pneumoniae (22.4%) and Staphylococcus aureus (15.5%). 57.2% of the Gram-negative bacteria and 48.7% of the Gram-positive isolates were resistant to ciprofloxacin. In the earlier (2009) screening, 39% of Gram-negative rods and 48% of Gram-positive cocci were indifferent to the drug.
A decrease in bacterial susceptibility of uropathogens to ciprofloxacin, a commonly prescribed drug in our population, is underlined, occurring possibly due to overuse pressure. Empirical initial treatment with ciprofloxacin would be inadequate in more than half of UTI cases, thereby counseling increased C/S testing of urines to provide existing sensitivity data for apt drug prescription.
PMCID: PMC3536602  PMID: 23268858
Urine; Isolates; Ciprofloxacin; Karachi

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