Recurrent urinary tract infection due to co-infection with extended spectrum b-lactamase-producer uropathogenic Escherichia coli and enteroaggregative E . coli

Case presentation: A 73-year-old woman with diabetes mellitus type 2 presented with fever, nausea, vomiting, burning sensation, painful and frequent urination. She was diagnosed with recurrent UTI (RUTI) due to co-infection with pansensitive enteroaggregative E. coli (EAEC) and MAR extended spectrum b-lactamase (ESBL)-producing uropathogenic E. coli (UPEC) and treated with azithromycin and levofloxacin (each for 10 days). Unfortunately, she did not respond. Plasmid profile analysis showed that the MAR E. coli strain harboured multiple plasmids including ,38 and ,80 MDa plasmids. PCR for detection of b-lactamase genes showed that the isolate was negative for blaTEM, blaSHV, blaOXA and blaCTX-M. To the best of our knowledge, this is the first report of co-infection with an EAEC and MAR ESBL-producer UPEC in Bangladesh.


Introduction
Urinary tract infections (UTIs) are frequent bacterial infections worldwide and the most commonly encountered bacterial infection, particularly in women.It is estimated that ,150 million cases of UTI occur worldwide, as many as 50 % of women and 12 % of men experience at least one symptomatic UTI during their lives and ,25 % of affected women have recurrent UTI (RUTI) (Foxman, 2010).Non-pathogenic E. coli are widely distributed in nature and an important part of the normal flora in the human gastrointestinal tract.E. coli causing UTI are classified as uropathogenic E. coli (UPEC); these strains account for 75-90 % of uncomplicated UTIs but are also an important aetiological agent of bacteraemia, sepsis and meningitis (Russo & Johnson, 2000;Yamamoto, 2007).
There are 14 serogroups of UPEC that are most frequently found in UTIs and 75 % of UTIs are caused by serogroups O4, O6, O14, O22, O75 and O83 (Li et al., 2010).O2, O4, O6 and O75 are the most common serogroups causing UTIs worldwide (George & Manges, 2010).Outbreaks due to particular UPEC strains have been reported from different parts of the world (George & Manges, 2010), indicating that these strains are capable of causing infections in the elderly and the immunosuppressed, including people with diabetes, HIV and chronic heart disease (Foxman, 2010).UPECs have distinct virulence factors, including adhesins, toxins, cytolysins, siderophores and surface components, that enable them to colonize and invade the urinary tract (Foxman, 2010).Colonization can lead to development of a quiescent intracellular reservoir in the bladder.Activation of this quiescent intracellular reservoir results in RUTIs (Anderson et al., 2003).
The increasing incidence of UTIs due to extended spectrum b-lactamase (ESBL)-producing E. coli is an emerging threat and of great concern throughout the Abbreviations: EAEC, enteroaggregative Escherichia coli; ESBL, extended spectrum b-lactamase; MAR, multiple-antibiotic-resistant; UTI, urinary tract infection; RUTI, recurrent UTI; UPEC, uropathogenic E. coli.
globe (Foxman, 2010).In addition, the ESBL-producing strains are also acquiring resistance to other, non-b-lactam antibiotics, which limits the empiric choice of treatment (Falagas et al., 2010;Foxman, 2010).However, limited data are available on UTIs caused by co-infection with ESBLproducing UPEC and enteroaggregative Escherichia coli (EAEC) in older people with diabetes in Bangladesh.
It is important that all health-care professionals should be aware of co-infection with multiple-antibiotic-resistant (MAR) UPEC and EAEC owing to their potential for lifethreatening complications.Here, we present a case report of an elderly woman with RUTI due to mixed infection with EAEC and MAR ESBL-producing E. coli.

Case presentation
A 73-year-old woman with diabetes mellitus type 2 and hypertension who came to Bangladesh on vacation with her family presented with fever, headache, fatigue, body ache, nausea, vomiting, abdominal pain, morning rise of temperature, shivering, polyuria, dysuria, pain in the pelvic area and back, and cloudy urine.Her past medical history was significant for recurrent UTIs for 15 years.She had a history of taking the following medications for the last 8 years: astatine (20 mg) (serastatin), lisinopril (5 mg), oxycodoneacetaminophen, calcium and gabapentin (300 mg).She also had insulin (8 units per day) for the last 3 years.
At the time of admission her blood pressure was 180/ 80 mmHg, her pulse rate was 80 beats min 21 , her respiration rate was 32 breaths min 21 , and her body temperature was 36.7 u C. Her abdomen was slightly tender but not distended.Microbiological tests of blood and urine are shown in Table 1.As the patient had a history of allergy to sulfonamides and trimethoprim, the antibiotic azithromycin (400 mg twice a day) was initially started after a urine sample had been taken for bacteriological analysis.Urine culture revealed lactose-fermenting colonies on MacConkey agar and biochemical tests showed properties of E. coli.Two types of colony, typical of EAEC with aaiC and aatA genes and of UPEC, were further confirmed using API 20E.
Antimicrobial sensitivity tests showed that the EAEC was pansensitive while the MAR UPEC strain was resistant to Abbreviations: HDL, high-density lipoprotein; hpf, high power field; LDL, low-density lipoprotein.
ampicillin, amoxicillin-clavulanate, cotrimoxazole, ciprofloxacin, ceftriaxone, cefixime and norfloxacin but susceptible to amikacin, azithromycin, gentamicin, mecillinam, imipenem, meropenem and nitrofurantoin.After 10 days of azithromycin treatment, a culture of urine was positive for only the MAR UPEC strain.At that point, levofloxacin (500 mg per day) was started and continued for 10 days.
Unfortunately, the patient also did not respond to levofloxacin.Several days after this, she flew home with her family.In a telephone call 1 month later, she conveyed that she had not recovered from the illness.She confirmed that she continued taking cranberry juice as medication and had started to use vaginal Vagifem (oestradiol) tablets to prevent vaginal dryness.

Discussion
E. coli is a very diverse species of bacteria found naturally in the intestinal tract of all humans and animals.Diarrheagenic E. coli are classified into six major pathotypes, each with distinct phenotypic and genetic make-up (Rasko et al., 2011).The diarrheagenic pathotypes of E. coli are also diverse in terms of disease potential and age and host specificity.E. coli can cause a range of human diseases, including UTI, neonatal meningitis and sepsis.UTIs are the most common form of extra-intestinal infections with E. coli.E. coli is the most common aetiology of UTIs and is responsible for 80-90 % of community-acquired UTIs.UPEC are different from the normal flora of E. coli in terms of better adaptability to living within the urinary tract and evading the host's immune response (Foxman, 2010).UPEC are very heterogeneous in nature.UPEC strains harbour several pathogenicity islands, which may encode adhesins, toxins, iron uptake systems, secretion mechanisms and capsules in order to enable them to develop successful UTI (Foxman, 2010).UPEC has multiple molecular pathways (for example, biofilm formation and urothelial cell invasion) that may contribute to pathogenesis.In addition, EAEC is the most recently identified diarrheagenic E. coli and is increasingly recognized as an emerging enteric pathogen that can cause infection in a wide range of age groups (Harrington et al., 2009;Huang et al., 2004).The annual incidence of EAEC infections ranges from 0.1 to 12 cases per 100 000 population.EAEC-mediated extra-intestinal disease was first reported by Erik and colleagues in 2012 (Boll, 2012).
EAEC has virulence factors that can cause symptomatic UTI (Herzog et al., 2013;Olesen et al., 2012).In Bangladesh, the true incidence of EAEC infections is unknown.UTI due to UPEC is likely to be underdiagnosed due to improper laboratory diagnostic methods and lack of awareness of the epidemiologic significance.UPEC can spread efficiently from person to person via intimate activity or faecal-oral transmission (Foxman, 2010).Transmission might differ in different environmental settings (Huppertz et al., 1997).
The first line of drugs to treat UTI includes ampicillin, cotrimoxazole and ciprofloxacin.However, emergence of resistance to these antimicrobial agents has complicated the situation and limits the empiric choice of treatment.The reported patient was allergic to cotrimoxazole; hence the treating physician initially prescribed her a macrolide antibiotic before doing culture and sensitivity tests.
Prescribing antibiotics prior to getting sensitivity test results is a common phenomenon in low-resource countries like Bangladesh.This contributes to the emergence of multiple antibiotic resistance among enteric pathogens (Larsson et al., 2000).A major concern regarding ESBL-producing E. coli is its high rate of co-resistance to non-b-lactam antibiotics, particularly quinolones, cotrimoxazole and aminoglycosides (Azap et al., 2010).In this study, the EAEC strain was uniformly sensitive to all tested antimicrobial agents whereas the UPEC showed resistance to ampicillin, ciprofloxacin, ceftriaxone and cefixime.The patient also did not adequately respond to fluoroquinolone (levofloxacin) therapy.When tested for antimicrobial drug susceptibility using the disc diffusion method with commercially available discs, two different types of E. coli isolate were recovered.The first type of isolate, a MAR ESBLproducing E. coli strain, was resistant to b-lactams, including ampicillin, ceftriaxone (MIC .64mg ml 21 ) and cefixime.The other type of isolate was a pansensitive EAEC.ESBL production was confirmed using a double disc diffusion test as described elsewhere (Ahmed et al., 2012).The isolates were also resistant to amoxicillin-clavulanate, cotrimoxazole, ciprofloxacin and norfloxacin, but susceptible to amikacin, azithromycin, gentamicin, mecillinam, imipenem, meropenem and nitrofurantoin.Although the patient's physician first prescribed azithromycin for 10 days and later levofloxacin for another 10 days, unfortunately the patient did not recover by the time she returned home.Alternative antibiotic choices for treatment of patients with ESBL-producing bacterial infection are limited.Carbapenems remain the most effective drugs of choice, but these antibiotics need to be administered by an intravenous or intramuscular route (DeBusscher et al., 2009).PCR specific for b-lactamase resistance genes of the ceftriaxone-resistant isolate showed that this strain was negative for bla TEM , bla SHV , bla OXA and bla CTX-M genes.This finding indicates that ESBL production in the MAR UPEC strain might be due to another class of genes.
Plasmid profile analysis of cefixime-resistant E. coli and all sensitive EAEC strains showed that both strains harboured an ,80 MDa plasmid which is common in E. coli (Talukdar et al., 2013).In addition, the multidrug-resistant UPEC strain also harboured plasmids 38, 3.4 and 2.5 MDa in size (Figure 1).These plasmids contribute to resistance to different classes of antibiotics (Talukdar et al., 2013).
The MAR E. coli strain persisted in the reported patient despite appropriate treatment with antimicrobial drugs.Thus, because of the long duration of carriage of this highly resistant strain, potential for dissemination to others is high.The infrequency of earlier reports on community-acquired ESBL-mediated infection in the United States does not suggest a high prevalence of these pathogens in the community.The wider distribution of ESBL-producer bacteraemia in patients visiting emergency departments has been reported by other investigators (Lee et al., 2012).Although regularly culturing urine samples for microbiological analysis is difficult, without continuous surveillance to detect and control ESBL-producer pathogens, the frequency of these pathogens can only be expected to increase.
The current lack of global routine surveillance systems for detecting EAEC and ESBL-producer UPEC, likely rendering the burden of these organisms under-reported, makes it difficult to address the extent to which these aetiological agents are causing extra-intestinal infection.To the best of our knowledge, our study is the first report of direct detection of co-infection of MAR ESBL-producer UPEC and EAEC due to RUTI in a diabetic patient in Bangladesh.Due to the high prescription of antibiotics for humans and livestock animals, antibiotic resistance has been increased in UPEC strains, and we recommend using antibiotics only in severe conditions and applying multi-antibiotic prescriptions.

Table 1 .
Biochemical analysis of blood sample and routine microscopic analysis of urine sample of the patient