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Prevalence of CTX-M beta-Lactamases in Escherichia coli from community-acquired urinary tract infections and associated risk factors among women in Cameroon

Background: In recent years, a worldwide dissemination of CTX-M beta-lactamase-type in Escherichia coli strains isolates from community-acquired urinary tract infections (CA-UTI) has been observed. However little is known on the prevalence and risk factors of this global threat in developing countries. Objective: The aim of this study was to study the prevalence and risk factors for CA-UTI in Yaoundé, Cameroon. Methods: Eighty six patients with urinary E.coli infection recruited from 10 health structures in the town of Yaoundé, Cameroon. After taking the first urine, faeces were collected from the patients for the study of the intestinal flora. The sample collection of faeces was done on a selected gel of enterobacteria resistant to third generation of cephalosporin. The molecular typing of extended-spectrum β-lactamase (ESBL) was carried out. Results: Eighty-six strains of E. coli from 86 patients were included. We found that 39 (45.3%) strains produced an extendedspectrum beta-lactamase. Among risk factors, previous use of antibiotic and the dry season were associated with the presence of an ESBL-producing strain in the urine. All ESBL were identified as CTX-M. The production of CTX-M was found to be significantly associated with resistance to fluoroquinolones, aminoglycosides and to the association of trimethoprim-sulfamethazole. Conclusions: The prevalence of CTX-M ESBL in Yaoundé, Cameroon, provides new evidence on the global dissemination of CTX-M and the extent of this phenomenon in developing countries.


INTRODUCTION
Urinary tract infections (UTIs) are a major public health problem in terms of morbidity and financial cost. [1] There are currently 150 million UTIs per year worldwide and this remains a major health problem in developing coun-tries. [2] These infections are mostly caused by bacteria and acquired in community. [3] Women are mostly affected. [4] Escherichia coli is responsible for 75%-90% of all urinary tract infections in community patients. Many studies of antimicrobial susceptibility of E. coli strains in urinary commu-nity patients were performed in industrialized countries. [3] The global spreading of CTX-M extended-spectrum betalactamase (ESBL) in these strains E.coli was observed and is been described as pandemic. [5] There is paucity of reports on ESBL in E. coli strains in community-acquired UTIs in developing countries. [2,[6][7][8] Few studies have been done on ESBL however they only looked at intestinal carriage. [9][10][11] The prevalence of CTX-M in E. coli strains responsible for community-acquired UTI in Cameroon or Central Africa has not been documented. The aim of our study was to determine the prevalence of bla CTX-M-type in E. coli strains in community-acquired urinary tract infections in women as well as its risk factors in Cameroon.

METHODS
From May 2011 to April 2012, we recruited all women who had UTI in 10 healthcare facilities (3 public hospitals and 7 laboratories in Yaoundé, Cameroon). Only those who had signed a consent form, filled and questionnaire on clinical data (history of UTIs, antibiotic use in the three preceding months, hospitalization in the last three months and the presence of burn) and demographical data and submitted a sample of stool were included. 86 smples from women with biological signs of E. coli UTI (leukocyturia > 10 5 leukocytes per ml of urine) and E. coli bacteriuria (> 10 5 UFC per ml of urine) were isolated in Cytine Lactose Electrolyte Deficient and identified by the API 20 E gallery (bioMérieux, Marcy l'Etoile, France). E. coli strains were stored in 1 ml of brain heart broth, supplemented with 10% glycerol and frozen at -80 • C until shipped together with the stool samples to the Bacteriology Laboratory of the Bichat-Claude Bernard Hospital, Paris, France were analyses were done.

Fecal antibiotic activity
Exposition to antibiotics at the time of fecal sampling was defined by the detection of fecal antibiotic activity. Fecal antibiotic activity was detected using a simple microbiological assay, performed as described elsewhere. [12] 2.3 PCR amplification This was done as decribed previously by Ruppé et al. [2] except that we used 1 colony and 1.7% agar gel on Sybergreen (see Table 1).

Statistical analysis
We used for analysis the R software, version 3.1.0. Risk factors were identified using univariate analysis with the Chi 2 or Fisher exact tests for qualitative variables of ANOVA for quantitative variables. All variables in the univariate analysis with a P-value less than .20 were introduced into a multivariate logistic model, using the backward strategy and testing some possible interactions. Some variables such as: the presence of antibiotics in stool and hospitalization in the past three months were systematically maintained in the model. Statistical differences with a p-value less than .05 were considered significant.

Ethics considerations
The Cameroon National Ethical Committee issued an ethical clearance (approval No. 207/CNE/SE/2011). After the main investigator had explained and read the project, all patients signed a consent form.

Population characteristics
We recruited a total of 86 patients in 3 government hospitals and seven laboratories. The median age was 33 years (IQR 22.75). Twelve patients (14%) had a history of urinary tract infection. The use of antibiotics at the time of sampling was found in about one third patients and 29.1% in the three months prior to inclusion. 12% of patients were hospitalized in the three months before the inclusion, and 11% of the patients had undergone surgery.
Eighty six E. coli strains were isolated. Among these, 74.4% of patients lived in the Center region, 11.6% were from the East, 10.5% from the West, 2.3% from the Littoral and 1.2% from the North West regions respectively.

Beta-lactamase characterization
All thirty nine strains of E. coli with an ESBL phenotype were analyzed. All ESBL producing E. coli strains were CTX-M of group 1. No strain of E.coli had TEM or SHV.

Risk factors associated with ESBL E. coli urinary tract infection
The mean age of patients with ESBL producing E. coli infection was 39.8 years against 36.7 years for patients with non-ESBL producing E. coli strain infection.
The univariate analysis (see Table 2) showed that factors associated with the occurrence of urinary tract infections were the dry season, the presence of a burn and presence of antibiotic in stool. Age of patients, previous hospitalizations for cases such as diabetes and kidney failure were not significantly associated with the occurrence of ESBL urinary tract infections.
The multivariate analysis as presented in Table 3

DISCUSSION
Our study is the first study on E. coli producing CTX -M in UTI among women in Cameroon. Although the number of acquired strains may be considered to be limited (n = 86), their level of antibiotic resistance is quite high regarding their community origin. As such, 39 strains of E. coli of community origin had an ESBL CTX-M.
Our study was conducted on the same target population and followed a similar protocol as those used in Senegal. The average prevalence found in this country was 6.5%. [15] Meanwhile the prevalence found in Cameroon was 45.3%. This result is higher than the Senegal study carried out 10 years ago.
In Cameroon, all E. coli strains isolated CTX-M are coresistant to other antibiotics families such as aminoglycosides, fluoroquinolones, trimethoprimsulfamethazole. A study in Dakar revealed low resistance rates of Enterobacteriaceae to tromethanolfosfomycin, a form of fosfomycin characterized by a high oral bioavailability. [16] Findings obtained in that study corroborates our results. Fosfomycin may be recommended as a reliable empirical treatment for simple urinary tract infections because of its ease in use (one dose), good tolerance and effectiveness. [17] The high prevalence of antibiotic resistance in the community would also be associated to the poor hygiene in Cameroon.
Since it is densely populated. During the dry season access to drinking water is a major problem. Water certainly promotes the circulation of Enterobacteriaceae strains (be they resistant or not). [18][19][20][21][22][23][24][25] Moreover, access to drinking water is not properly insured in many parts of the city. As such, Enterobacteriaceae from stools can easily affect many people through contaminated water. Many households get water from unhealthy streams used as a dumping ground by residents. A study carried out by De Boeck in Kinshasa (Central Africa ) revealed the presence of ESBL producing Enterobacteriaceae in drinking water [26] and another study revealed the presence of E. coli in rivers and wells. [21] In a village community in South Guyana, it was also proven that even in the absence of a strong antibiotic pressure, promiscuity promoted the circulation of resistant strains and thus increased the prevalence of carrying them. [27] The uncontrolled consumption of antibiotics may promote the selection of E. coli strains producing ESBL, which usually results in the empirical prescription for self-medication, poor adherence of patients to antibiotic therapy, the quality of antibiotics (generic under dosed) and the questionable preservation (street drugs). The prescription of antibiotics is in most cases dispensed without microbiological data, and is therefore based on clinical and also on the patient's ability to pay his/her antibiotics. This also applies to medical tests, which are non-refundable, and this is because Cameroon has not implemented the social security system.
In addition, most of these strains were co-resistant to fluoroquinolones, aminoglycosides and trimethoprimsulfamethazole, thus drastically reducing therapeutic options in the treatment of such infections.

CONCLUSION
The findings of this study show that the global dissemination of CTX-M-type ESBL affects particularly Cameroon and its capital city, since nearly half of the community strains of E. coli accounting for urinary tract infections had this type of ESBL. Furthermore, these strains were in most cases coresistant to other antibiotics widely used in E. coli infection treatments.
The situation of CTX-M in Cameroon he brings new facts to explain the current global pandemic. Hygiene and the normal use of antibiotics must be in the centre for the fight against the spread of such multidrug-resistant strains.
We believe it is right time authorities in Africa in general and Cameroon in particular should be concerned by providing health facilities structures to carry out complete bacteriological analysis, harmonize antibiogram procedures in order to detect bacteria producing extended spectrum betalactamases and to stop the use of antibiotics such as amoxicillin, ciprofloxacin and cotrimozaxole in empirical treat-ment since there's a strong resistance of Enterobacteriaceae to these antibiotics. Fosfomycin may be recommended as a reliable empirical treatment for urinary tract infections, unfortunately this antibiotic is not yet available in Cameroon.