Globally, there is an increasing occurrence of multidrug-resistant (MDR) Enterobacteriaceae with extended-spectrum β-lactamases (ESBLs) and/or plasmid-encoded AmpC (pAmpC) β-lactamases in clinical and environmental settings of significant concern. Therefore, we aimed to evaluate the occurrence of ESBL/pAmpC genetic determinants, and some essential non-β-lactam genetic determinants in the MDR phenotypic antimicrobial resistance in Enterobacteriaceae isolates recovered from hospital wastewater. We collected samples from two hospitals in Amathole and Chris Hani District Municipalities in the Eastern Cape Province, South Africa, within October and November 2017. Using the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF), we identified a total of 44 presumptive Enterobacteriaceae isolates. From this, 31 were identified as the targeted Enterobacteriaceae members. Thirty-six percent of these belonged toKlebsiella oxytoca, while 29% wereKlebsiella pneumoniae. The other identified isolates includedCitrobacter freundiiandEscherichia coli(10%),Enterobacter asburiae(6%),Enterobacter amnigenus,Enterobacter hormaechei, andEnterobacter kobei(3%). We established the antibiotic susceptibility profiles of these identified bacterial isolates against a panel of 18 selected antibiotics belonging to 11 classes were established following established guidelines by the Clinical and Laboratory Standard Institute. All the bacterial species exhibited resistance phenotypically against at least four antibiotic classes and were classified as MDR. Notably, all the bacterial species displayed resistance against cefotaxime, ampicillin, nalidixic acid, and trimethoprim-sulfamethoxazole. The generated multiple antibiotic resistance indices ranged between 0.5 to 1.0, with the highest value seen in oneK.oxytocaisolated. Molecular characterization via the Polymerase Chain Reaction uncovered various ESBLs, pAmpCs, and other non-β-lactam encoding genes. Of the phenotypically resistant isolates screened for each class of antibiotics, the ESBLs detected werebla_CTX-Mgroup (including groups 1, 2, and 9) [51.6% (16/31)],bla_TEM[32.3% (10/31)],bla_OXA-1-like[19.4% (6/31)],bla_SHV[12.9% (4/31)],bla_PER[6.5% (2/31)],bla_VEB[3.2% (1/31)],bla_OXA-48-likeandbla_VIM[15.4% (2/13)], andbla_IMP[7.7% (1/13)]. The pAmpC resistance determinants detected werebla_CIT[12.9% (4/31)],bla_FOX[9.7% (3/31)],bla_EBC[6.5% (2/31)], andbla_DHA[3.2% (1/31)]. The frequencies of the non-β-lactam genes detected werecatII[79.2% (19/24)],tetA[46.7% (14/30)],sulIandsulII[35.5% (11/31)],tetB[23.3% (7/30)],aadA[12.9% (4/31)],tetC[10% (3/30)], andtetD[3.3% (1/30)]. These results indicate that hospital wastewater is laden with potentially pathogenic MDR Enterobacteriaceae with various antibiotic resistance genes that can be spread to humans throughout the food chain, provided the wastewaters are not properly treated before eventual discharge into the environment.

The increasing occurrence of multidrug-resistant Enterobacteriaceae in clinical and environmental settings has been seen globally as a complex public health challenge, mostly in the developing nations where they significantly impact freshwater used for a variety of domestic purposes and irrigation. This paper detail the occurrence and antibiogram signatures of the Enterobacteriaceae community in Tsomo and Tyhume rivers within the Eastern Cape Province, the Republic of South Africa, using standard methods. The average distribution of the presumptive Enterobacteriaceae in the rivers ranged from 1 × 10² CFU/100ml to 1.95 × 10⁴ CFU/100ml. We confirmed 56 (70.8%) out of 79 presumptive Enterobacteriaceae isolated being species within the family Enterobacteriaceae through the Matrix-Assisted Laser Desorption Ionization Time of Flight technique. Citrobacter-, Enterobacter-, Klebsiella species, and Escherichia coli were selected (n = 40) due to their pathogenic potentials for antibiogram profiling. The results of the antibiotic susceptibility testing gave a revelation that all the isolates were phenotypically multidrug-resistant. The resistance against ampicillin (95%), tetracycline and doxycycline (88%), and trimethoprim-sulfamethoxazole (85%) antibiotics were most prevalent. The Multiple Antibiotic Resistance indices stretched from 0.22 to 0.94, with the highest index observed in a C. freundii isolate. Molecular characterisation using the PCR technique revealed the dominance of bla_TEM (30%; 12/40) among the eight groups of β-lactams resistance determinants assayed. The prevalence of others was bla_CTX-M genes including group 1, 2 and 9 (27.5%), bla_SHV (20%), bla_OXA-1-like (10%), bla_PER (2.5%), and bla_VEB (0%). The frequencies of the resistance determinants for the carbapenems were bla_KPC (17.6%), bla_GES (11.8%), bla_IMP (11.8%), bla_VIM (11.8%), and bla_OXA-48-like (5.9%). Out of the six plasmid-mediated AmpC (pAmpC) genes investigated bla_ACC, bla_EBC, bla_FOX, bla_CIT, bla_DHA, and bla_MOX, only the first four were detected. In this category, the most dominant was bla_EBC, with 18.4% (7/38). The prevalence of the non-β-lactamases include tetA (33.3%), tetB (30.5%), tetC (2.8%), tetD (11.1%), tetK (0%), tetM (13.9%), catI (12%), catII (68%), sulI (14.3%), sulII (22.9%) and aadA (8.3%). Notably, a C. koseri harboured 42.8% (12/28) of the genes assayed for which includes five of the ESBL genes (including the only bla_PER detected in this study), two of the pAmpC resistance genes (bla_ACC and bla_CIT), and five of the non-β-lactamase genes. This study gives the first report on C. koseri exhibiting the co-occurrence of ESBL/AmpC β-lactamase genes from the environment to the best of our knowledge. The detection of a bla_PER producing Citrobacter spp. in this study is remarkable. These findings provide evidence that freshwater serves as reservoirs of antimicrobial resistance determinants, which can then be easily transferred to human beings via the food chain and water.

The aquatic environments play a critical function in the widespread of antibiotic-resistant bacteria, ultimately impacting human health. We evaluated the abundance of Enterobacteriaceae and the various resistance gene determinants in final effluents of wastewater treatment plants (WWTPs) in the Eastern Cape Province, Republic of South Africa. A total of 44 presumptive Enterobacteriaceae was recovered following standard isolation methods. Upon molecular confirmation through Matrix-Assisted Laser Desorption/Ionization Time of flight (MALDI TOF), a high predominance of Klebsiella pneumoniae (24%) was noted. All the confirmed isolates (n = 29) subjected to a panel of eighteen antibiotics categorized into eleven different classes were multidrug-resistant (MDR). They displayed resistance against antibiotics in more than three different classes. Notably, one E. cloacae exhibited resistance against all the antibiotics assayed. The multiple antibiotic resistance indices (MARI) ranged from 0.22 to 1.0, indicating an environment with high pressure of antibiotics. Conventional Polymerase Chain Reaction (PCR) analysis showed that 72.4% of the isolates harboured at least one β-lactamase genetic determinant. The most predominant extended-spectrum β-lactamases were bla_CTX-M-group (21.5%), bla_TEM (20%), and bla_SHV (16.9%) while the most predominant plasmid-mediated AmpC-type β-lactamases were bla_CIT and bla_ACC (25.9%) and bla_EBC (22.2%). A total of 86.2% of the Enterobacteriaceae harboured a minimum of one non-β-lactam resistance gene determinant with predominance observed in catII (25%), sulII (15.8%), and sulI (14.5%). These results demonstrate an abundance of multidrug-resistant (MDR) Enterobacteriaceae in WWTPs’ effluents. The study confirms the need to optimize current WWTPs’ processes to improve the quality of effluents and ultimately reduce the potential risks of using such water when discharged into the environment.

The high prevalence of infections arising from Klebsiella species is related to their ability to acquire and disseminate exogenous genes associated with mobile genetic elements such as integrons. We assessed the prevalence, diversity, and associated gene cassettes (GCs) of integrons in Klebsiella species. The isolates recovered from wastewater and hospital effluents, rivers, and animal droppings were identified using the conventional Polymerase Chain Reaction (PCR) with primers targeting the gryA, pehX, and 16S–23S genes. The antimicrobial resistance profile and the Extended-Spectrum and Metallo β-lactamases production were carried out using standard microbiological techniques. PCR, DNA sequencing analyses, and Restriction Fragment Length Polymorphism were used to characterize the integrons and their associated GCs. Furthermore, the genotypic relationships between the different isolated K. pneumoniae were determined using Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR. About 98% (51/52) of the confirmed isolates harboured an integrase gene, with 80% intI1, while the remaining 20% concurrently harboured intI1 and intI2, with no intI3 observed. About 78% (40/51) of the bacterial strains were positive for a promoter, the P2R2, investigated, while 80% (41/51) harboured at least one of the qacEΔ1 and sul1. Three different GCs arrangements identified were aac(6′)-Ib, aadA1-dfrA1, and dfrA1-sat2. At a similarity index of 60%, the ERIC-PCR fingerprints generated were categorized into nine clusters. Our study is the first to reveal the features of integrons in Klebsiella spp. recovered from environmental sources in the Eastern Cape Province, South Africa. We conclude that the organisms' sources are repositories of integrons harbouring various gene cassettes, which can be readily mobilized to other microorganisms in similar or varied niches.

The increasing occurrence of multidrug-resistant Enterobacteriaceae in clinical and environmental settings has been seen globally as a complex public health challenge, mostly in the developing nations where they significantly impact on freshwater used for a variety of domestic purposes and irrigation. In this paper, we give details as regards the occurrence and antibiogram signatures of Enterobacteriaceae community in Tsomo and Tyhume rivers within the Eastern Cape Province, Republic of South Africa using standard methods. Average distribution of the presumptive Enterobacteriaceae in the rivers ranged from 1 × 10 ² CFU/100ml to 1.95 × 10 ⁴ CFU/100ml. We confirmed 56 (70.8%) out of 79 presumptive Enterobacteriaceae isolated being species within the family Enterobacteriaceae through the Matrix-Assisted Laser Desorption Ionization Time of Flight technique. From this, Citrobacter -, Enterobacter -, Klebsiella species and Escherichia coli were selected (n=40) due to their pathogenic potentials for antibiogram profiling. The results of the antibiotic susceptibility testing gave a revelation that all the isolates were phenotypically multidrug-resistant while resistance against ampicillin (95%), tetracycline and doxycycline (88%) and trimethoprim-sulfamethoxazole (85%) antibiotics were most prevalent. The Multiple Antibiotic Resistance indices stretched from 0.22 to 0.94, with the highest index observed in a C. freundii isolate. Molecular characterisation using the PCR technique revealed the dominance of bla _TEM (30%; 12/40) among the ten groups of extended-spectrum β-lactamase (ESBL) genes assayed. The prevalence of others was bla _CTX-M genes including group 1, 2 and 9 (27.5%), bla _SHV (20%), bla _KPC (17.6%), bla _GES (11.8%), bla _IMP (11.8%), bla _VIM (11.8%), bla _OXA-1-like (10%), bla _PER (2.5%), bla _OXA-48-like (5.9%) and bla _VEB (0%). From the six plasmid-mediated AmpC (pAmpC) genes investigated bla _ACC , bla _EBC , bla _FOX , bla _CIT , bla _DHA and bla _MOX , only the first four were detected. In this category, the most dominant was bla _EBC, with 18.4% (7/38). The prevalence of the non-β-lactamases include tetA (33.3%), tetB (30.5%) , tetC (2.8%), tetD (11.1%), tetK (0%), tetM (13.9%), catI (12%), catII (68%), sulI (14.3%), sulII (22.8%) and aadA (8.3%). Notably, a C. koseri harboured 42.8% (12/28) of the genes assayed for which includes five of the ESBL genes (including the only bla _PER detected in this study), two of the pAmpC resistance genes ( bla _ACC and bla _CIT ) and five of the non-β-lactamase genes. To the best of our knowledge, this study gives the first report on C. koseri exhibiting co-occurrence of ESBL/AmpC β-lactamase genes from the environment. The detection of a bla _PER producing Citrobacter spp. in this study is remarkable. These findings provide evidence that freshwater serves as reservoirs of antimicrobial resistance determinants which can then be easily transferred to human beings via the food chain and water.

One strategy for combating antimicrobial resistance in many infections is to combine antibacterial compounds to create combinations that outperform each molecule alone. In this study, we examine and study the inhibitory effect of combining two drugs belonging to different antibiotic classes to obtain a possible potentiating effect against some Enterobacteriaceae isolates harbouring integrons recovered from rivers and effluents of hospital and wastewater treatment plants in Eastern Cape Province, South Africa. These integrons could easily enable the isolates to acquire genes that confer additional resistance against conventional antibiotics. The minimum inhibitory concentration of the various antibiotics was determined using the broth microdilution, while the checkerboard method was used to determine the fractional inhibitory concentration indices (FICIs). A total of 26.3% (10/38) of the interactions were categorised as synergistic, while 73.7% (28/38) were indifferent. None of the combinations were antagonistic. The time–kill assays revealed all the synergistic interactions as bactericidal. Therefore, the combinations of gentamicin with tetracycline, ciprofloxacin, and ceftazidime against multidrug-resistant (MDR) Klebsiella pneumoniae, tetracycline–ceftazidime combination against MDR Escherichia coli, colistin combinations with ceftazidime and gentamicin, and tetracycline–gentamicin combinations against MDR Citrobacter freundii may be future therapeutic alternatives. Hence, the synergistic combinations reported in this study must be assessed further in vivo before their clinical applications.