Study design and setting
A prospective, cross sectional study conducted between the periods January to June 2019. The study site was Umzikantu Red Meat Abattoir, Zimbane Mthatha, Mthatha River and Mthatha dam. Umzikantu Red Meat Abattoir is a high throughput abattoir located in Zimbane location in Mthatha, Eastern Cape. It is the only operational red meat abattoir serving Mthatha and its environs in the O.R Tambo District Municipality. It is certified and have the capacity to slaughter 50 units of animals on a daily basis. One unit equals to one cow/ox or two calves or six sheep or four pigs. The abattoir is open to the public and offers slaughter and cutting services at an affordable price. It also doubles as a wholesaler supplying meat to butcheries, supermarkets and hospitals.
Mthatha Dam (31°33′2″S 28°44′24″E Coordinates) is an earth-fill type dam on the Mthatha River, located close to Mthatha Town, in the OR Tambo District Municipality of the Eastern Cape. This dam was founded in 1977 to serve municipal and industrial purposes. The Department of Water and Sanitation oversees the affairs of the dam. The catchment area of the dam is 886 km2 with surface area measuring 25.42 km2. It has a height of 38 m with the length measuring 620 m. The reservoir capacity of the dam is 253, 674, 000 m3.
Sample collection
Abattoir environment samples: Using standard methods, one hundred (100) mililitres of abattoir effluents were taken from two sampling points into sterile bottles appropriately labelled. All samples were stored in cooler boxes for transportation to the Medical Microbiology laboratory at Walter Sisulu University Mthatha, for further analyses, within 4 h of sample collection.
Aquatic environment samples: Water samples from the Mthatha dam were collected aseptically in sterile 100mL Duran Schott glass bottles from different sampling points by directly dipping the bottles about 20cm below the surface of the water. After collection, the samples were placed in iced cooler boxes, transported to the laboratory and kept at about 4oC until analysed.
Bacteriological analysis
Membrane filtration method was used for isolation and enumeration according to standard methods [65]. For all samples, three volumes of 100mL were filtered through 0.45 𝜇m pore sized gridded membrane filter (Whatman Laboratory Division, Maidstone, England) using a water pump (model Sartorius 16824). Filter was removed and aseptically placed on CHROMagarTM Pseudomonas (CHROMagarTM; Paris, France) plates ensuring that no air bubbles were trapped. All media were prepared according to the manufacturers’ instructions (CHROMagarTM; Paris, France). Each sample was analysed in triplicate. Effluent samples from Abattoir were serially diluted and 1mL of the 5 fold serial dilutions was filtered through the 0.45 𝜇m pore sized gridded membrane filter (Whatman Laboratory Division, Maidstone, England) using a water pump (model Sartorius 16824) before being placed aseptically on to the CHROMagarTM Pseudomonas (CHROMagarTM; Paris, France) agar plates. The plates were incubated aerobically at 370C for 24-48 hours. Blue colonies characteristics of Pseudomonas spp. were enumerated.
Characterization of Pseudomonas aeruginosa
Blue colonies typical of Pseudomonas species on the chromogenic medium were subcultured on both Cetrimide agar and CHROMagar to get pure colonies. Characteristics grape-like odour was a useful marker of identification. Phenotypic tests such as Gram stain, oxidase test and catalase test were performed [66]. Species identification was carried out using Gram-negative ID type 2 panels (Beckman Coulter, Inc. USA) of MicroScan autoScan-4 automated system (Dade Behring Inc., Deerfield, IL). Growth at 420C [66] in an aerobic incubator was also used to confirm the identity of the P. aeruginosa isolates.
Antimicrobial susceptibility testing
Antimicrobial susceptibility testing was performed by MicroScan autoScan-4 system using Microscan dehydrated broth microdilution negative MIC Panel Type 44 (NM44) (Beckman Coulter, Inc. USA) following the manufacturer’s guidelines [67]. The following antibiotics were tested in the panels: amikacin, aztreonam, cefepime, ceftazidime, ciprofloxacin, doripenem, gentamicin, imipenem, levofloxacin, meropenem, piperacillin/tazobactam, piperacillin and tobramycin. MICs were interpreted following CLSI guidelines [68]. ATCC Quality control organisms used were P. aeruginosa ATCC 27853 and Escherichia coli ATCC 25922. Non-susceptibility included the combination of resistance and intermediate resistance. MDR P. aeruginosa was defined as non-susceptibility to at least one agent in three or more antimicrobial categories according to Magiorakos et al. [69].
Multiple antibiotic resistance (MAR) index: MAR was calculated and interpreted for the isolates as described by Gufe et al. [59]. Briefly, it is described as the ratio of the number of antibiotics to which isolates were resistant (a) to the total number of antibiotics to which the isolates were exposed (b) i.e: MARI = a/b. The MARI of individual P. aeruginosa from the two different sources was calculated. Bacteria having MARI (>0.2) originate from high risk sources of contamination where several antibiotics have been used, while, MARI value of ≤0.2 indicates strains from sources where antibiotics have seldom or never been used.
Molecular confirmation of strains by rPCR
DNA Extraction: DNA was extracted from overnight colonies of bacterial culture grown on Cetrimide agar. This was re-suspended in Roche Magna Pure Bacteria Lysis Buffer, vortexed briefly, heated at 95°C for 10 minutes and pelleted by centrifugation at 13000 g for 10 minutes. Four hundred microliters were used as a specimen in the MagNa Pure Compact System (Roche Applied Science, Indianapolis), using MagNa Pure Compact Nucleic Acid isolation kit 1 according to manufacturer’s instructions. Elution tubes containing 200 µl purified nucleic acids were stored at – 800C until further use. The LightCycler 2.0 instrument (Roche Applied Science, Germany) and Fast start LightCycler 480 Hybprobes Master kit (Roche Diagnostics, USA) were used for polymerase reaction. Specific primers targeting the gene, species-specific gyrB were amplified by singleplex rPCR using primers and probes designed by TIB Molbiol, Germany. 16S rRNA analysis was also carried out to confirm identity of the isolates.
Molecular ESBL and MBL detection by rPCR
Real-time PCR for blaCTX-M, blaSHV, blaTEM, blaIMP and blaVIM. Isolated P. aeruginosa colonies on Cetrimide agar and CHROMagar Pseudomonas were selected for genomic DNA extraction. Template DNA was extracted by MagNA Pure Compact (MPC) using MPC Nucleic Acid isolation kit according to manufacturer’s instruction. Real time PCR was carried out in the LightCycler 2.0 instrument (Roche Applied Science, Germany) using Fast start LightCycler 480 Hybprobes Master kit (Roche Diagnostics, USA). Specific primers and probes (Table 3) targeting the genes CTX-M, SHV, TEM, IMP and VIM were amplified by singleplex rPCR. Primers were designed by TIB-Molbiol (Berlin, Germany). rPCR assay was performed in a 32 carousels using 20 𝜇L capillaries with each capillary containing a total volume of 20 𝜇L; including 2𝜇L of LightCycler FastStart DNA Master Hybridization Probes (Roche Diagnostics), 2 𝜇L of primers (0.5mM for each forward and reverse), 2 𝜇L of probe, 2.4 𝜇L of MgCl2, 2 𝜇L of extracted DNA, and water to make up the volume of 20 𝜇L. The rPCR protocol are as shown in Table 4.
Table 3: Primer sequences for detection of blaCTX-M, blaSHV, blaTEM, blaIMP,blaVIM genes and gyrB
Target gene
|
Primers
|
Primers sequences (5’-3’)
|
Tm in 00C
|
References
|
blaCTX-M
|
CTX-M forward primer
|
ATGAGYACCAGTAARGTKATGGC
|
58.7
|
70
|
|
CTX-M reverse primer
|
ATCACKCGGRTCGCCIGGRAT
|
59.3
|
|
|
CTX-M Probe
|
FAM-CCCGACAGCTGGGAGACGAAACGT-BBQ
|
70.2
|
|
blaSHV
|
SHV forward primer
|
TCCCATGATGAGCACCTTTAAA
|
56.8
|
71
|
|
SHV reverse primer
|
TCCTGCTGGCGATAGTGGAT
|
58.6
|
|
|
SHV Probe
|
FAM-TGCCGGTGACGAACAGCTGGAG-BBQ
|
68.3
|
|
blaTEM
|
TEM forward primer
|
GCATCTTACGGATGGCATGA
|
56.6
|
71
|
|
TEM reverse primer
|
GTCCTCCGATCGTTGTCAGAA
|
57.7
|
|
|
TEM Probe
|
FAM-CAGTGCTGCCATAACCATGAGTGA-BHQ1
|
62.2
|
|
blaIMP
|
IMP forward primer
|
GGGCGGAATAGAGTGGCTTA
|
57.6
|
72
|
|
IMP reverse primer
|
GGCTTGAACCTTACCGTCTTTTT
|
59.3
|
|
|
IMP Probe
|
FAM-CGATCTATCCCCACGTATGCATCTGAATTAACA-BHQ1
|
67.4
|
|
blaVIM
|
VIM forward primer
|
TGCGCTTCGGTCCAGTAGA
|
59.0
|
72
|
|
VIM reverse primer
|
TGACGGGACGTATACAACCAGAT
|
58.5
|
|
|
VIM Probe
|
FAM-CTTCTATCCTGGTGCTGCGCATTCG-BHQ1
|
67.6
|
|
gyrB
|
gyrB forward primer
|
CCT GAC CAT CCG TCG CCA CAA
|
|
73
|
|
gyrB reverse primer
|
CGC AGC AGG ATG CCG ACG CC
|
|
|
|
gyrB probe
|
6-FAM-CCG TGG TGG TAG ACC TGT TCC CAG ACC-BHQ
6-FAM-CCG TGG TGG TAG ACC TGT TCC CAG ACC-BBQ
|
|
This study
|
Table 4 Real-time PCR cycle protocol
Protocol
|
Temperature
|
Acquisition mode
|
Time
|
Ramp rate
|
Cycle
|
Denaturation
|
950C
|
None
|
5 minutes
|
4.4
|
1
|
Quantification: annealing
|
950C
|
None
|
30 seconds
|
4.4
|
45
|
Extension
|
600C
|
Single
|
1 minute
|
4.4
|
|
No third step
|
|
Cooling
|
400C
|
None
|
30 seconds
|
4.4
|
1
|
Absolute quantification was carried out using the Light Cycler software 4.05. Data were obtained during annealing period. Fluorescence was measured once every cycle immediately after the 60°C incubation (extension step). Fluorescence curves were analyzed with the LightCycler software, version 4.05. Results were expressed by determination of threshold cycle (Ct) value which signified the cycle at which sample fluorescence became remarkably different from baseline signal. Positive control strains used included Klebsiella pneumoniae ATCC 51503 (blaCTX-M), Klebsiella pneumoniae ATCC 700603 (blaSHV), Escherichia coli NCTC 13351 (blaTEM), P. aeruginosa NCTC 13437 (blaVIM) and Escherichia coli NCTC 13476 (blaIMP). These were obtained from the National Institute of Communicable Diseases (NICD), Johannesburg, South Africa.