Study population and settings
This study was conducted in a rural, district and urban, tertiary hospital, encoded for ethical reasons as H1 and H2, respectively, during 2 months from May 2017 to June 2017 in uMgungundlovu district, South Africa. The district hospital (H1) represents the smallest level of hospital and provides four services including obstetrics and gynaecology, paediatrics and child health, general surgery and general medicine with 141 beds. In contrast, the tertiary hospital (H2) offers several specialties, receives referral patients according to a nationally agreed referral plan and has approximately 505 beds.
Patient enrolment and questionnaire survey
Total sampling was performed for the recruitment of participants i.e. all patients older than 18 years old, hospitalized in medical or surgical ward of the hospitals H1 and H2, and willing to participate were included in the study. Oral and written informed consent was obtained from all study participants after explanation of the procedure and purpose of the study. Patient information was gleaned from questionnaires completed by patients and data from patient records. Information was codified prior to analysis to maintain confidentiality.
Sample collection took place in both surgical and general medical wards during a two-month period, 1 month at each of the hospitals. Rectal swabs that were collected aseptically with Amies swabs from symptomatic in-patients, at three-time points, at admission, after 48 h and at discharge (whenever possible) formed the carriage sample. Isolates from symptomatic patients originating from tissue, blood, urine, intravenous catheters, and sputum routinely processed in the microbiological laboratory during the sampling period formed the clinical sample.
Definitions of terms
The specimen (blood, urine, sputum, tissue, intravenous catheter tips, fluid/aspirate and superficial swab) collected for diagnostic purpose from a symptomatic hospitalized patient was considered clinical sample. The clinical isolates were recovered from clinical samples obtained from patients hospitalized in various units of the selected hospitals. In contrast, carriage sample was the rectal swab collected from hospitalized patients at different time-points (admission, after 48 h and at discharge) out of diagnostic tests performed at hospitals.
Identification of gram-negative ESKAPE bacteria
During the sample collection, all rectal swabs were cultured onto MacConkey agar with and without cefotaxime (2 mg/L). After incubation for 18-24 h at 37 °C, each morphotype growing on MacConkey with cefotaxime (MCA + CTX) was subjected to Gram staining, catalase and oxidase tests, followed by biochemical identification with API 20E (bioMérieux, Marcy l’Etoile, France) and Vitek® 2 System (bioMérieux, Marcy l’Etoile, France) using the GN card according to the manufacturer’s instructions. Pure colonies were stored into Tryptone Soya Broth supplemented with 30% glycerol at − 20 °C for future use.
All growing colonies were phenotypically screened for ESBL, AmpC, KPC, MBL, and OXA-48 production using ROSCO DIAGNOSTICA (Taastrup, Denmark) using 0.5 McFarland on Mueller-Hinton agar according to the manufacturer’s instructions.
Antimicrobial susceptibility testing
Minimum inhibitory concentrations (MICs) were determined via broth microdilution for all presumptive ESBLs and/or AmpCs, and/or MBL producers. Ampicillin, cefoxitin, cefuroxime, cefotaxime, ceftazidime, meropenem, imipenem, ertapenem, amikacin, gentamicin, ciprofloxacin, tigecycline, tetracycline, doxycycline, nitrofurantoin, and colistin constituted the antibiotic panel for carriage isolates. The Vitek® 2 System and Vitek® 2 Gram-negative Susceptibility card (AST-N255) were used to determining the MICs of clinical isolates. The results of MIC tests were interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints  and the MDR which is the resistance to three or more classes of antibiotics was also assessed. Escherichia coli ATCC 25922, K. pneumoniae ATCC 700603 and K. pneumoniae ATCC 51503 were used as controls.
Genomic DNA of selected strains were extracted using GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Genomic DNA was stored at − 20 °C for future use.
Multiplex polymerase chain reaction (M-PCR)
The isolates were subjected to molecular testing using conventional and M-PCR assays to identify blaCTX-M group 8/25 (blaCTX-M-gp8/25), blaSHV, blaTEM, blaOXA-1-like, blaOXA-48, blaKPC, blaVIM, blaIMP and blaGES genes as previously described  (Additional file 1: Table S1).
Real-time polymerase chain reaction (RT-PCR)
RT-PCR was performed to ascertain blaAmpC, blaCTX-M-group-1 (blaCTX-M-gp1), blaCTX-M-group-2 (blaCTX-M-gp2) and blaCTX-M-group-9 (blaCTX-M-gp9) resistance genes. Results were analysed on a programmable automate QuantStudio5™ (Applied Biosystems, CA, USA) using the Taqman Universal Master Mix 2× (Applied Biosystems, CA, USA) and ready-made assays (Thermo Scientific, CA, USA). Thermal temperature running conditions were as follows: UNG activation at 50 °C for 2 min, initial denaturation at 95 °C for 10 min, 30 cycles of denaturation at 95 °C for 10 s, annealing/extension at 60 °C for 1 min and a final extension at 60 °C for 30 s. The results were interpreted with QuantStudio™ design and analysis software version 1.4 (Applied Biosystems, CA, USA).
Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR) was used to establish the link of different strains within and between hospitals, wards, carriage and clinical samples as well as across sampling points. The primers ERIC1 5’ATGTAAGCTCCTGGGGATTCAC3’ and ERIC2 5’AAGTAAGTGACTGGGGTGAGCG3’  were used and PCR reactions were carried out in a 10 μl volume containing 5 μl of DreamTaq Green Polymerase Master Mix 2X (Thermo Fisher Scientific, Johannesburg, South Africa), 2.8 μl of nuclease free water, 0.1 μl of each primer (100 μM), and 2 μl of DNA template. The reactions were carried out with the following cycling conditions: initial denaturation at 94 °C for 3 min, 30 cycles consisting of a denaturation step at 94 °C for 30 s, annealing at 50 °C for 1 min, extension at 65 °C for 8 min, a final extension step at 65 °C for 16 min and final storage at 4 °C. The generated amplicons were resolved by horizontal electrophoresis on 1.5% (wt/vol) Tris-Borate-EDTA (Merck, Germany) agarose gels together with the Quick-load®1-kb (Biolabs, New England) and run in an electric field of 110 V for 2 h 30 min. Electrophoresis gels were visualized by a UV light trans-illuminator, images were captured using a Gel Doc™ XR+ system (BioRad Laboratories, CA, Foster City, USA) and analysed by Image Lab™ Software (version 4.0, BioRad Laboratories, CA, Foster City, USA).
ERIC-PCR profiles were normalized using the Quick-load®1-kb (Biolabs, New England) DNA molecular weight marker as the external standard. For cluster analysis, data were exported to Bionumerics software (version 7.6, Applied Maths, TX, USA). Strains were allocated to different clusters by calculating the similarity coefficient from the homology matrix using the Jaccard method. Dendrograms were constructed based on the average linkages of the matrix and using the Unweighted Pair-Group Method (UPGMA). Optimization and band tolerance were set at 1% (version 7.6, Applied Maths, TX, USA) and 80% similarity cut-off was used to define clusters.
Data was coded and entered on an Excel spreadsheet (Microsoft Office 2016) and analysed using STATA (version 14.0, STATA Corporation, TX, USA). Risk factors for ESBL-mediating MDR Gram-negative ESKAPE colonization were ascertained by univariate and multivariate logistic regression analyses. Prevalence of MDR carriage was compared between categories (viz. hospital, ward and time-point) using the chi-square and Fisher’s exact test as appropriate. A p-value < 0.05 was regarded as statistically significant.