Between 1992 and 2010, our laboratory at the KEMRI Centre for Microbiology Research received 912 E. coli isolates from 13 health centres in Kenya. All the 912 isolates were resistant to penicillins alone (e.g. ampicillin), or a combination of penicillins and different classes of β-lactam antibiotics. These isolates were from urine (395), blood (202), stool (315) and were obtained from confirmed cases of urethral tract infections (UTIs), septicaemia and diarrhoea-like illnesses respectively. Out of the 912 isolates, 255 (28 %) were obtained between 1992 and 1999 while 657 (72 %) were obtained between 2000 and 2010. This difference was as a result of an increase in isolation rates as a result of better detection and screening techniques in recent years. These isolates were obtained from 350 patients seeking outpatient treatment and 562 were from hospitalised patients. Upon receipt, the isolates were sub-cultured on MacConkey agar (Oxoid, Basingstoke, U`K) and species identification done using standard biochemical tests as described before . Ethical clearance to carry out this study was obtained from the KEMRI/National Ethics Committee (Approval: SSC No. 1177).
Antimicrobial susceptibility profiles
Antimicrobial susceptibility tests were performed for all the 912 isolates using antibiotic discs (Cypress diagnostics, Langdorp, Belgium) on Mueller Hinton agar (Oxoid, Basingstoke, United Kingdom). E. coli ATCC 25922 was included as a control strain on each test occasion. Susceptibility tests were interpreted using the Clinical and Laboratory Standards Institute (CLSI) guidelines . The antibiotics included in this panel were: - ampicillin (AMP, 10 μg), oxacillin (OXA, 30 μg), amoxicillin (AML, 30 μg ), cephalothin (KF, 30 μg), cefuroxime (CXM 30 μg), cefotaxime (CTX, 30 μg) and ceftazidime (CAZ, 30 μg). Other antibiotics included cefepime (FEP, 30 μg), aztreonam (AZT, 30 μg), and cefoxitin (FOX, 30 μg). β-lactam/β-lactamase inhibitor combinations included amoxicillin/clavulanic acid (AMC, comprising amoxicillin 20 μg and clavulanic acid 10 μg), ampicillin/sulbactam (AMS) combinations in rations of 20 μg and 10 μg respectively, and piperacillin/tazobactam (TZP) in potency ratio of 100/10 μg respectively. Imipenem (IM 30 μg) was used to test susceptibility to carbapenems.
Detection and Interpretation of β-lactamase phenotype
Two strategies were used for detection of β-lactamase phenotypes as detailed in the CLSI guidelines , and in other related studies . The first strategy was the double-disc synergy test (m-DDST) in which the β-lactam antibiotics were placed adjacent to the amoxicillin/clavulanic (AMC) disc at inter-disc distances (centre to centre) of 20 mm. A clear extension of the edge of the disc zones towards the AMC (ghost zones or zones of synergy) was interpreted as positive for ESBL production. In the combined disc method (CDM), tests were first done using β-lactam antibiotics and then repeated using discs containing combinations of β-lactam/β-lactamase inhibitors. A result indicating a ≥ 5 mm increase in zone diameter for the β-lactam/β-lactamase inhibitor disc was interpreted as production of ESBLs [45, 46]. The results from the m-DDST and CDM methods were also used for empiric categorization of strains into NSBL-, IRT-, ESBL- CMT- and pAmpC-like β-lactamase phenotypes as detailed before .
PCR detection of β-lactamase genes
Preparation of DNA used as template in PCR reactions was obtained by boiling bacteria suspension from an 8 hr culture at 95 °C for 5 minutes. The supernatant was stored at -20o C until further use. Subsequent PCR amplifications were carried out in a final volume of 25 μL or 50 μL. A minimum of 5 μL of template DNA and 1 μL of 10 mM concentration of both forward and reverse primers were used in PCR reactions. Isolates from our collection that had been found to carry various bla genes in past studies [24, 27, 47], were used as positive controls in PCR screening for genes of interest. Sterile distilled water or E. coli strains susceptible to all β-lactam antibiotics were used as negative controls. PCR products were analyzed using electrophoresis in 1.5 % agarose gels and stained with ethidium bromide. Visualization of the PCR products was done under UV light and the image recorded with the aid of a gel documentation system (Bio-Rad Laboratories, Hercules, CA, USA).
Selection of isolates for further analysis
Isolates from each phenotype were selected for further analysis using PCR and sequencing strategies. For phenotypes with a high number of isolates (i.e. more than a hundred strains), at least 56% of the isolates were selected for further analysis. In order to minimize bias, the isolates selected from each phenotype were proportion to the total number of isolates obtained during each year of isolation (1992 to 2010). Similarly, the number of isolates selected from urine, stool and blood specimen was proportional to the total number of strains isolated from each specimen-type obtained from both hospitalized and non-hospitalized patients. Using this criterion, 586 (64%) of the 912 isolates were selected for further analysis. Regardless of the source phenotype, all the selected isolates were investigated for carriage of the complete panel of bla genes screened for in this study.
Screening for bla genes
The strains were screened for genes frequently reported among members of family Enterobacteriaceae . The list of primers used is indicated in Table 4. Consensus primers published in past studies were used for screening for bla
SHV and bla
TEM[48, 49], bla
CTX-M and bla
CMY. Isolates positive using bla
CTX-M consensus primers were screened using primers specific for CTX-M group I to IV as described in a previous study . Isolates positive using the bla
CMY primers were analyzed using primers for bla
CMY-1-like and bla
CMY-2-like genes . Detection of other β-lactamase genes was done as previously described for bla
OXA-like [53, 54], bla
PER-like  , bla
ACC-like , bla
VEB-like , and bla
DHA-like genes .
Amplicons used as template in sequencing reactions were purified using the QIAquick PCR purification kit (Qiagen Ltd., West Sussex, UK). Bi-directional sequencing of the products was done using the DiDeoxy chain termination method in ABI PRISM 310 automatic sequencer (PE Biosystems, Foster City, CA, USA). Consensus primers were used for sequencing except for bla
genes that were sequenced using group-specific primers. Translation of nucleotide sequences was done using bioinformatics tools available at the website of the National Center of Biotechnology Information on http://www.ncbi.nlm.nih.gov. Alignment of the translated enzyme amino acid sequence was done against that of the wild-type using the ClustalW program on http://www.ebi.ac.uk . Identification of enzyme mutations at amino acid level was determined by comparing the translated amino acid sequence with that of the wild-type enzyme published at http://www.lahey.org/studies.