https://orcid.org/0000-0001-6100-9090
Figures
Abstract
Carbapenem-resistant bacteria (CRB) present a significant global public health concern. Sub-Saharan Africa has borne a heavy burden of CRB with a reported prevalence of up to 60% in some patient populations. es in Africa focus on clinical CRB isolates, with limited data on their spread in the natural environment. Therefore, the purpose of this study was to report the recovery of CRB from Nairobi River surface waters and nearby anthropogenic and zoonotic sources in Nairobi County, Kenya. A total of 336 CRB were recovered from 336 (250 mL) samples, with 230 of the samples (68.5%) producing one or more CRB isolates. CRB were recovered most commonly from untreated sewage influent (100% of 36 samples; 79 total isolates), treated effluent (93% of 118 samples; 116 total isolates), Nairobi River surface waters upstream (100% of 36 samples; 57 total isolates), downstream (100% of 36 samples; 45 total isolates), and way downstream from the wastewater treatment plant (73% of 11 samples; 19 total isolates), slaughterhouse effluent discharges 1.5%, (5/336), animal contact areas 0.9%, (3/336), a manhole sewer from the affluent neighborhood of Karen at 2.7%, (9/336) respectively. The CRB included Escherichia coli (158, 47%), Klebsiella pneumoniae (74, 22%), and Enterobacter spp (43, 13%). Aeromonas spp (29, 9%) Acinetobacter baumannii (12, 3.6%), Citrobacter freundii (7, 2.1%), Pseudomonas aeruginosa (5, 1.5%) and other species (8, 2.4%). CRB genotypes included blaNDM (246, 73.2%), blaKPC (40, 12%), blaVIM (51, 15.2%), blaOXA-48-like (65, 19.3%), blaIMP (15, 4.5%), and blaGES (7, 2.1%). Sixty-nine of the CRB isolates (20.5%) harbored multiple carbapenemase-encoding genes. Our results indicate that clinically important CRB are commonly present in Nairobi River surface water and from nearby wastewater and livestock sources. These pose an important public health threat that requires urgent intervention strategies and additional investigation.
Citation: Too RJ, Kariuki SM, Gitao GC, Bebora LC, Mollenkopf DF, Wittum TE (2024) Carbapenemase-producing bacteria recovered from Nairobi River, Kenya surface water and from nearby anthropogenic and zoonotic sources. PLoS ONE 19(11): e0310026. https://doi.org/10.1371/journal.pone.0310026
Editor: Dhammika Leshan Wannigama, Chulalongkorn University Faculty of Medicine and King Chulalongkorn Memorial Hospital, THAILAND
Received: July 2, 2024; Accepted: August 22, 2024; Published: November 14, 2024
This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Data Availability: All relevant data are within the manuscript.
Funding: This research was supported by the Government of Kenya through KEMRI Internal Research Grant number KEMRI/IRG/EC0012. The funders had no role in study design, data collection, and analysis, the decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Carbapenem antimicrobials are generally reserved for use as a last option for the treatment of invasive multidrug-resistant bacterial infections in high-risk patients [1]. The emergence of carbapenem-resistant bacteria (CRB) represents an important public health threat that has been recognized by both the US Centers for Disease Control (CDC) [2] and the World Health Organization (WHO) [3]. CRB poses an important threat to high-risk patient populations, such as those housed in intensive care units, where case fatality rates as high as 50% have been reported [4]. In addition, healthcare environments including medical devices, equipment carts, wheelchairs and gurneys, and a wide variety of other surfaces may become contaminated with CRB and serve as fomites for transmission [4, 5]. Bacterial mechanisms of carbapenem resistance can include restriction of membrane permeability and cellular target alteration, but carbapenemase is the mechanism of primary epidemiologic importance. This is because carbapenemase production is frequently encoded on mobile genetic elements that are readily transferred within and between bacterial species. Individual carbapenemase genotypes that initially emerged regionally, including blaNDM in Asia, blaKPC in North America, and blaVIM in Europe have subsequently disseminated globally. This global spread of CRB is not limited to healthcare environments, and healthy individuals in the community may become colonized following exposure to a variety of anthropogenic or zoonotic sources. These sources may include retail food products [6–9], agricultural [10–12] or companion animals [11–13], wastewater effluent [16, 17], or the natural environment including wildlife [18] and surface waters [9–22]. The epidemiology of CRB in the environment, including their main points of origination and impact, has not been fully elucidated. CRB has been reported in untreated wastewater influent, treated wastewater effluent, and surface waters near wastewater treatment plant discharge [17, 23–28], in many regions of the world, but data for Africa are not available. The prevalence of CRB among clinical isolates recovered from patients in Sub-Saharan Africa has been reported to be as high as 60% [29, 30]. A variety of globally disseminated CRB genotypes including blaKPC, blaNDM, blaVIM, blaOXA-48-like, and blaIMP from Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii have been reported from various hospitals in East Africa, where CRB prevalence ranged from 22.4 to 34% [31–33]. However, the frequency that CRB escapes the healthcare environment and disseminates in surface waters in Africa is unknown.
We hypothesized that CRB present in human and animal waste in Nairobi County, Kenya routinely disseminate into the Nairobi River where they pose a risk to human and animal health. This study aims to report the frequency and recovery of CRB in the environment from Nairobi River surface water and from potential sources of dissemination including a wastewater treatment plant, a neighborhood sewer, and animal agriculture facilities in Nairobi County, Kenya.
Materials and methods
Description of study design and study area
This was a longitudinal study design where we systematically sampled multiple sites in the vicinity of Nairobi, Kenya including; the Ruai wastewater treatment plant (WWTP) located at: 1° 18’0” South, 36° 55’0” East [34], the Nairobi River near the WWTP effluent discharge sites, the Dagoretti slaughterhouse located at 00°4’7’S, 109° 11’41”E including its animal contact areas and wastewater treatment effluent, as well as influent and effluent from a manhole sewer in a nearby affluent neighborhood (Karen). The Ruai WWTP treats about 85,000 m3/day which represents approximately 80% of the wastewater generated from Nairobi County. The Dagoretti slaughterhouse serves as an economic hub for the people of Nairobi and its environs, where meat slaughtered from the abattoir is transported to Nairobi city and other nearby towns in Kenya. Different samples comprising raw untreated WWTP sewage influent, WWTP discharge final effluent, fecal samples from the animals grazing at the WWTP area, Nairobi River surface water at sites upstream, downstream, way downstream from the WWTP, slaughterhouse discharges and Swiffer samples from the wiped surfaces on animal contact areas including holding pens, hallways, and killing floor, cradles and hooks were aseptically collected once a week between the months of April 2021 and April 2022.
Sampling strategy, sample collection, handling, and transportation
A systematic sampling technique was used to collect wastewater samples (250 mL) every Thursday of the week from the Ruai WWTP raw untreated influent and three fully treated effluent points (Effluent A, Effluent B, and Effluent C). Surface water samples (250 mL) were obtained from the Nairobi River at sites located upstream, downstream, and way downstream from WWTP effluent discharge sites at a distance of 100 meters apart all from the effluent discharge sites. Swiffer samples were collected from livestock stunning pens (lairage), hooks, killing areas, cradles, drainages, and other animal contact hallways at the slaughter facility. Samples of slaughterhouse effluent were collected from a drainage channel before discharge into the Kavuthi stream (a tributary of the Nairobi River). All samples were collected aseptically to avoid contamination and packaged in well-labeled sterile whirl-pak bags then placed into an ice cooler box and transported to the Kenya Medical Research Institute (KEMRI) Microbiology laboratory for processing.
Sample processing and laboratory procedures
Filtration of water samples.
To isolate CRB from water samples including influent and effluent, a membrane filter technique was used as previously described [34, 35]. The raw water samples collected in 250 mL polypropylene bottles were recorded and filtered through multiple cellulose membrane filters (Thermo Fischer Scientific™ Nalgene™ Filter Membranes) of different pore sizes to allow all the waters to pass through to avoid clogging utilizing a Millipore vacuum filtration system apparatus; starting with 41 μm filter paper, 20 μm, 10 μm, 1.2 μm 0.8 μm, and culminating with a 0.45 μm filter to capture bacteria [36]. All filters for a single sample were then placed into the same whirl-pak bag for culture.
Culture and isolation of carbapenemase-producing bacteria.
Fifty mL of MacConkey (MAC) broth (BD) modified with 0.5 μg/mL meropenem and 70 μg/mL ZnSO4 (Oxoid Ltd) was added into whirl-pak bags containing filter papers used for filtration or/ and Swiffers used to wipe surfaces and fecal samples (5 g) which were then incubated aerobically for 18–24 hours at 37°C. After overnight growth, the suspension was inoculated onto MacConkey agar plates modified with 0.5 μg/mL meropenem and 70 μg/mL zinc sulfate and incubated for 18–24 hours at 37°C [37–39]. Based on morphological characteristics of Gram-negative bacteria and lactose fermentation, up to four pink colonies were picked for isolation from each sample plate and inoculated onto Mueller Hinton agar (MHA) modified with meropenem 0.5 μg/mL and 70 μg/mL ZnSO4 and incubated at 37°C for 24 hours [37]. Carbapenemase production was confirmed using the CarbaNP test [37] and matrix-assisted laser desorption/ionization (MALDI) time of flight (TOF) analyzer mass spectrometry (MS) was used to identify bacterial species where a MALDI target plate was spotted with a small amount of bacterial cell colony in the middle of both wells for that isolate ID with a toothpick. After putting all samples on each of their respective wells, 1μL of the bacterial test standard (BTS) on just the BTS well for quality control then dried at room temperature for a few minutes. Later the sample spot was overlayed with 1μL of formic acid onto each well (including the BTS well) and the plate was left to dry for several minutes. Matrix solution (10 mg/mL cyno-4-hydroxycinnamic acid in acetonitrile-water-trifluoroacetic acid (TFA) was vortexed for 3 minutes then, 1μL of the matrix liquid was put onto each well (including the BTS well) then left to dry prior to analysis.
Identification of CRB using the Carba-NP test directly from bacterial cultures.
For Carba-NP, colonies from MHA agar were scraped off with a 1μL loop and suspended in two 1.5 mL Eppendorf tubes containing 100 μL of 20 mM Tris-HCl lysis buffer / Solulyse™ solution pH 7.8 already standardized then mixed [37, 40–42]. Standard control strains carrying blaKPC, and blaNDM-1 were included and incubated for 2 hours at 37°C based on the hydrolysis of the β-lactam ring of imipenem (Sigma) [37]. Thereafter, 15% glycerol stock was prepared from culture broths and Carba-NP positive isolates were preserved at -80°C for further analyses.
Detection of carbapenemase-encoding genes
DNA extraction.
Bacterial DNA was extracted from CRB isolates by creating boiled lysates as previously described [43]. Isolates were emulsified in 200 μL nuclease-free water in a 1.5 mL Eppendorf tube placed in a hot plate at 100°C for 10 minutes and centrifuged at 16,000 rpm for 3 minutes to obtain the DNA template which was stored at -20°C until further analysis.
Control strains and primer sequences.
The presence of specific carbapenemase-encoding genes including blaKPC, blaNDM, blaIMP, blaOXA-48-like, blaGES, and blaVIM was determined using PCR for all phenotypic CRB isolates. Standard control strain (ATCC®BAA-1705™ Klebsiella pneumoniae ART 2008133 [D-05, 1338] blaKPC+blaNDM) was used as a positive control. The primer sequences were selected based on the specificity and sensitivity of the PCR product size distinguishable by agarose gel electrophoresis in multiplex PCR systems from different studies, described earlier [44–46] (Table 1).
Multiplex PCR for CRB genotype.
Amplification reactions were performed [43, 47, 48] at a reaction volume of 25 μL. Three μL of template DNA was added to a master mix containing PuReTaq™ Ready-To-Go™ PCR beads (Cytiva, Global Life Sciences Solutions, Marlborough, MA, USA), 1.5 μL of forward and reverse primers, and 19 μL of nuclease-free water to a final volume of 25 μL.
The cycling conditions for all the PCR amplifications of carbapenem-resistant genes were as follows: 94°C for 5 minutes for initial denaturation and 30 cycles of 95°C for 30 seconds (denaturation), annealing at 52°C for 1 minute (for all primers except for blaKPC which was at 58°C) and 72°C for 1 minute 30 seconds (extension), followed by 72°C for 10 minutes (final extension); performed in 0.2 mL Eppendorf PCR tubes in a thermal cycler (MJ Research PTC-200). Amplified PCR products were separated and visualized by electrophoresis using a 1% agarose tris-acetate-EDTA gel containing 0.5 μg EtBr staining. A 100–1,000 bp DNA ladder (Fermenters) was used to determine molecular weight.
Data management and statistical analysis.
Using R- software version 4.2.1, data were analyzed using descriptive statistics using frequencies and percentages of CRB recovery computed as proportions of all samples collected and isolates recovered and presented using frequencies and percentages.
Ethical considerations.
Study approval was sought from the Center Scientific Committee, Center for Microbiology Research (CSC-CMR), and Scientific Ethical Review Unit (SERU)-KEMRI protocol number: KEMRI/SERU/CMR/0126/4092. The National Commission for Science Technology and Innovation (NACOSTI) identification number 522511 and License No: NACOSTI/P/20/7287, and the Faculty Committee at the Department of Veterinary Pathology, Microbiology and Parasitology of the University of Nairobi. This was an environmental study and no consent was required. Other Approvals to access the field sites were sought from the Nairobi water and sewerage company (NWSC) and the County Government of Kiambu Livestock, Fisheries and Veterinary Services.
Results
Identification and characterization of CRB in the environment
A total of 336 samples were collected during the study period, of which 157 were from the WWTP, 83 were from Nairobi River surface water, 74 were from the slaughterhouse, and 22 were from the affluent neighborhoods of Karen manhole sewer (Table 2). From these 336 samples, 230 (68.5%) yielded 336 CRB isolates, with some samples yielding as many as four phenotypically distinct CRB (Table 2). Samples from the WWTP influent and effluent were most likely to contain CRB (94%) while Swiffer samples from the slaughterhouse animal contact surfaces had the lowest frequency of CRB recovery (2%) (Table 2).
Frequency and distribution of CRB in study sites
Of the 336 CRB isolates, 158 (47.0%) were identified as Escherichia coli, 74 (22%) as Klebsiella pneumoniae, 43 (12.8%) as Enterobacter spp, 29 (8.6%) as Aeromonas spp., 12 (3.6%) as Acinetobacter baumannii, 7 (2.1%) as Citrobacter freundii, 5 (1.5%) as Pseudomonas aeruginosa and 8 (2.4%) were other species including Plesiomonas shigelloides, Citrobacter brakii, Kluyvera cryocrescens, Klebsiella vericola, Wautersiella falsenii, Acinetobacter bereziniae, and Camamonas kerstersii.
Frequency and distribution of carbapenemase-encoding genes
blaNDM either alone or with other carbapenemase encoding genes was the most frequently identified CRB genotype (n = 246, 73.2%), and was recovered from all the sampling sites. blaOXA-48-like was present in 65 isolates (19.3%), blaVIM was present in 51 isolates (15.2%), blaKPC was present in 40 isolates (11.9%) blaIMP was present in 15 isolates (4.5%), and blaGES was present in 7 isolates (2.1%) (Table 3).
Some isolates had multiple carbapenemase genes present.
blaNDM alone or together with other carbapenemase genes was present in 85.4% of E. coli, 67.6% of K. pneumoniae, 41.9% of E. bugandensis, 31% of A. caviae, and 36.4% of C. freundii and all other species. The distribution of CRB genotypes among the various bacterial species is summarized in Table 4.
Sixty-nine of the CRB isolates (20.5%) harbored multiple carbapenemase-encoding genes (Table 5). The majority (41, 59.4%) of these isolates carried 2 genes, while (22, 31.9%) carried 3, and (6, 8.7%) carried 4 carbapenemase-encoding genes (Table 5). The most frequently observed combination CRB genotype was blaNDM + blaVIM carried by eleven (15.9%) isolates (Table 5).
Discussion
We observed a high frequency of CRB from environmental samples from surface waters of the Nairobi River, and from nearby sources including Ruai WWTP influents and effluents, and Dagoretti slaughterhouse discharges that yielded multiple bacterial species expressing both phenotypic and genotypic carbapenem resistance, our results are consistent with other studies [39, 52, 53] where environmental samples showed 88% and 50% phenotypically confirmed carbapenemase-producing strains. We identified CRB producing a variety of different carbapenemase enzymes detected from wastewater and swiffer samples from different environmental niches.
The organisms that we recovered included Enterobacter spp., K. pneumoniae, A. baumannii, and P. aeruginosa, which the WHO had designated as being of critical importance. However, we also recovered E. coli and Aeromonas spp., which have been reported in hospitals, WWTPs, farms, and other environments [24, 53, 54]. While the majority of scientific literature focuses on clinical CRB isolates, E. coli is the most frequently recovered CRB from environmental samples as a result of being a conserved bacteria [55], and its ability to exchange resistance genes via horizontal gene transfer [56].
The blaNDM genotype was the most frequently detected among the CRB. The majority of the E. coli and some K. pneumoniae isolates carried blaNDM alone or co-harbored with other carbapenemase genes. This result is not unexpected because Africa accounts for approximately 10.8% of all blaNDM-1 cases reported worldwide [57]. The most common carbapenemase enzymes reported in clinical isolates across all African countries are NDM-type and OXA-type [30]. blaNDM was first reported in Kenya in 2007, originally from K. pneumoniae and later from an A. baumannii hospital outbreak [31, 58]. However, there is a lack of data on CRB from environmental sources in Kenya including surface water. Previous reports of clinical CRB in Kenya identified K. pneumoniae and E. coli harboring blaNDM and blaOXA alleles [59, 60]. Our results suggest that in Kenya, CRB has spread from hospital settings into the environment as has been reported in other countries [26].
Among the CRB, K. pneumoniae and E. coli have been highlighted by the WHO in their priority list of pathogens [61, 62]. Numerous studies have demonstrated the international spread of CRB genotypes, most commonly blaKPC and blaNDM. In Egypt [63], clinical CRB isolates harboring blaOXA-48-like have been reported. In addition, others [24, 54, 64] have reported CRB harboring blaOXA-48-like, blaNDM, and blaKPC in environmental niches, including WWTP in Eastern Cape Province, South Africa. A similar observation of a low prevalence of blaNDM-1, blaNDM-7, and blaOXA-181-producing E. coli clinical isolates in Iran has been reported [65]. Our results emphasize the potential importance of environmental CRB in Sub-Saharan Africa and suggest the need for greater research in this area.
It is noteworthy that 2475 tons of trash are created in Nairobi daily, and the majority of it is dumped directly into the Nairobi River from both industrial activity and informal settlements located around Nairobi County [66]. Consequently, the Nairobi River is heavily contaminated with untreated industrial and human garbage. This waste may be a source of chronic selective pressure exerted by antibiotics and other chemical pollutants including heavy metals such as lead and mercury [67]. Prior research [68–71] has primarily focused on antimicrobial residues in wastewater and water surfaces. Our results suggest that the dissemination of antimicrobial-resistant bacteria including CRB in the natural environment of Kenya, including the Nairobi River, is an important public health threat as well. Recent studies have reported clinical CRB similar to the CRB in our study [1, 33, 72, 73]. This suggests that hospitals and other healthcare settings may be the original source of the CRB recovered from the natural environment. Furthermore, the presence of CRB in the manhole sewer of the affluent neighborhoods of Karen and from the slaughterhouse suggests community transmission of CRB. This is an important public health concern for Kenya and all of Africa.
In summary, the presence of a wide variety of CRB from the Nairobi River in Kenya, and from nearby anthropogenic and zoonotic sources of contamination provides evidence that CRB originating in healthcare environments can disseminate in the community and into natural environments. Our research demonstrates the ability of environmental and wastewater AMR surveillance to identify epidemiologically important strains and clinically relevant antimicrobial resistance genes in Africa. Based on this information, control measures can be taken to reduce the risk to public health including proper waste treatment, both onsite at healthcare facilities and the final effluents from WWTPs before they are discharged into the receiving environment.
Acknowledgments
We would like to thank and acknowledge the Kenya Medical Research Institute (KEMRI), The Ohio State University (OSU) College of Veterinary Medicine, and The University of Nairobi (UON) for guidance, infrastructure, and laboratory supplies.
References
- 1. Jeong S, Lee N, Park MJ, Jeon K, Kim HS, Kim HS, et al. Genotypic Distribution and Antimicrobial Susceptibilities of Carbapenemase-Producing Enterobacteriaceae Isolated From Rectal and Clinical Samples in Korean University Hospitals Between 2016 and 2019. Ann Lab Med [Internet]. 2022 Jan 1 [cited 2024 Jun 22];42(1):36–46. Available from: http://annlabmed.org/journal/view.html?doi=10.3343/alm.2022.42.1.36
- 2.
Centers for Disease Control and Prevention (U.S.). Antibiotic resistance threats in the United States, 2019 [Internet]. Centers for Disease Control and Prevention (U.S.); 2019 Nov [cited 2024 Jun 23]. Available from: https://stacks.cdc.gov/view/cdc/82532
- 3.
World Health Organization. WHO report on surveillance of antibiotic consumption: 2016–2018 early implementation [Internet]. Geneva: World Health Organization; 2018 [cited 2024 Jun 23]. 113 p. Available from: https://iris.who.int/handle/10665/277359
- 4. Mariappan S, Sekar U, Kamalanathan A. Carbapenemase-producing Enterobacteriaceae: Risk factors for infection and impact of resistance on outcomes. Int J App Basic Med Res [Internet]. 2017 [cited 2024 Jun 22];7(1):32. Available from: https://journals.lww.com/10.4103/2229-516X.198520 pmid:28251105
- 5. Dankittipong N, Fischer EAJ, Swanenburg M, Wagenaar JA, Stegeman AJ, De Vos CJ. Quantitative Risk Assessment for the Introduction of Carbapenem-Resistant Enterobacteriaceae (CPE) into Dutch Livestock Farms. Antibiotics [Internet]. 2022 Feb 21 [cited 2024 Jun 22];11(2):281. Available from: https://www.mdpi.com/2079-6382/11/2/281 pmid:35203883
- 6. Galler H, Feierl G, Petternel C, Reinthaler FF, Haas D, Grisold AJ, et al. KPC-2 and OXA-48 carbapenemase-harbouring Enterobacteriaceae detected in an Austrian wastewater treatment plant. Clinical Microbiology and Infection [Internet]. 2014 Feb [cited 2024 Jun 22];20(2):O132–4. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1198743X14600616 pmid:24033741
- 7. Abdallah HM, Alnaiemi N, Reuland EA, Wintermans BB, Koek A, Abdelwahab AM, et al. Fecal carriage of extended-spectrum β-lactamase- and carbapenemase-producing Enterobacteriaceae in Egyptian patients with community-onset gastrointestinal complaints: a hospital -based cross-sectional study. Antimicrob Resist Infect Control [Internet]. 2017 Dec [cited 2024 Jun 22];6(1):62. Available from: http://aricjournal.biomedcentral.com/articles/10.1186/s13756-017-0219-7
- 8. Liu BT, Song FJ. Emergence of two Escherichia coli strains co-harboring mcr-1 and blaNDM in fresh vegetables from China. IDR [Internet]. 2019 Aug [cited 2024 Jun 22];Volume 12:2627–35. Available from: https://www.dovepress.com/emergence-of-two-escherichia-coli-strains-co-harboring-mcr-1-and-bland-peer-reviewed-article-IDR
- 9. Brouwer MSM, Tehrani KHME, Rapallini M, Geurts Y, Kant A, Harders F, et al. Novel Carbapenemases FLC-1 and IMI-2 Encoded by an Enterobacter cloacae Complex Isolated from Food Products. Antimicrob Agents Chemother [Internet]. 2019 Jun [cited 2024 Jun 22];63(6):e02338–18. Available from: https://journals.asm.org/doi/10.1128/AAC.02338-18 pmid:30910900
- 10. Mollenkopf DF, Stull JW, Mathys DA, Bowman AS, Feicht SM, Grooters SV, et al. Carbapenemase-Producing Enterobacteriaceae Recovered from the Environment of a Swine Farrow-to-Finish Operation in the United States. Antimicrob Agents Chemother [Internet]. 2017 Feb [cited 2024 Jun 22];61(2):e01298–16. Available from: https://journals.asm.org/doi/10.1128/AAC.01298-16 pmid:27919894
- 11. He T, Wei R, Zhang L, Sun L, Pang M, Wang R, et al. Characterization of NDM-5-positive extensively resistant Escherichia coli isolates from dairy cows. Veterinary Microbiology [Internet]. 2017 Aug [cited 2024 Jun 22];207:153–8. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0378113517305461 pmid:28757017
- 12. Wang J, Yao X, Luo J, Lv L, Zeng Z, Liu JH. Emergence of Escherichia coli co-producing NDM-1 and KPC-2 carbapenemases from a retail vegetable, China. Journal of Antimicrobial Chemotherapy [Internet]. 2018 Jan 1 [cited 2024 Jun 22];73(1):252–4. Available from: http://academic.oup.com/jac/article/73/1/252/4259082 pmid:29029065
- 13. Daniels JB, Chen L, Grooters SV, Mollenkopf DF, Mathys DA, Pancholi P, et al. Enterobacter cloacae Complex Sequence Type 171 Isolates Expressing KPC-4 Carbapenemase Recovered from Canine Patients in Ohio. Antimicrob Agents Chemother [Internet]. 2018 Dec [cited 2024 Jun 22];62(12):e01161–18. Available from: https://journals.asm.org/doi/10.1128/AAC.01161-18 pmid:30249699
- 14. Cole SD, Rankin SC. Characterization of 2 Klebsiella pneumoniae carbapenemase–producing Enterobacterales isolated from canine rectal swabs. J VET Diagn Invest [Internet]. 2022 Mar [cited 2024 Jun 22];34(2):306–9. Available from: http://journals.sagepub.com/doi/10.1177/10406387211065501
- 15. Köck R, Daniels-Haardt I, Becker K, Mellmann A, Friedrich AW, Mevius D, et al. Carbapenem-resistant Enterobacteriaceae in wildlife, food-producing, and companion animals: a systematic review. Clinical Microbiology and Infection [Internet]. 2018 Dec [cited 2024 Jun 22];24(12):1241–50. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1198743X18303392 pmid:29654871
- 16. Nasri E, Subirats J, Sànchez-Melsió A, Mansour HB, Borrego CM, Balcázar JL. Abundance of carbapenemase genes (blaKPC, blaNDM and blaOXA-48) in wastewater effluents from Tunisian hospitals. Environmental Pollution [Internet]. 2017 Oct [cited 2024 Jun 22];229:371–4. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0269749117307133 pmid:28614760
- 17. Mathys DA, Mollenkopf DF, Feicht SM, Adams RJ, Albers AL, Stuever DM, et al. Carbapenemase-producing Enterobacteriaceae and Aeromonas spp. present in wastewater treatment plant effluent and nearby surface waters in the US. Zhou Z, editor. PLoS ONE [Internet]. 2019 Jun 26 [cited 2024 Jun 22];14(6):e0218650. Available from: https://dx.plos.org/10.1371/journal.pone.0218650 pmid:31242271
- 18. Ballash GA, Dennis PM, Mollenkopf DF, Albers AL, Robison TL, Adams RJ, et al. Colonization of White-Tailed Deer (Odocoileus virginianus) from Urban and Suburban Environments with Cephalosporinase- and Carbapenemase-Producing Enterobacterales. Elkins CA, editor. Appl Environ Microbiol [Internet]. 2022 Jul 12 [cited 2024 Jun 22];88(13):e00465–22. Available from: https://journals.asm.org/doi/10.1128/aem.00465-22
- 19. Hamza D, Dorgham S, Ismael E, El-Moez SIA, Elhariri M, Elhelw R, et al. Emergence of β-lactamase- and carbapenemase- producing Enterobacteriaceae at integrated fish farms. Antimicrob Resist Infect Control [Internet]. 2020 Dec [cited 2024 Jun 22];9(1):67. Available from: https://aricjournal.biomedcentral.com/articles/10.1186/s13756-020-00736-3
- 20. Zurfluh K, Hächler H, Nüesch-Inderbinen M, Stephan R. Characteristics of Extended-Spectrum β-Lactamase- and Carbapenemase-Producing Enterobacteriaceae Isolates from Rivers and Lakes in Switzerland. Appl Environ Microbiol [Internet]. 2013 May [cited 2024 Jun 22];79(9):3021–6. Available from: https://journals.asm.org/doi/10.1128/AEM.00054-13
- 21. Sivalingam Poté, Prabakar . Environmental Prevalence of Carbapenem Resistance Enterobacteriaceae (CRE) in a Tropical Ecosystem in India: Human Health Perspectives and Future Directives. Pathogens [Internet]. 2019 Oct 2 [cited 2024 Jun 22];8(4):174. Available from: https://www.mdpi.com/2076-0817/8/4/174 pmid:31581701
- 22. Tanner WD, VanDerslice JA, Goel RK, Leecaster MK, Fisher MA, Olstadt J, et al. Multi-state study of Enterobacteriaceae harboring extended-spectrum beta-lactamase and carbapenemase genes in U.S. drinking water. Sci Rep [Internet]. 2019 Mar 8 [cited 2024 Jun 22];9(1):3938. Available from: https://www.nature.com/articles/s41598-019-40420-0 pmid:30850706
- 23. Woodford N, Ellington M, Coelho J, Turton J, Ward M, Brown S, et al. Multiplex PCR for genes encoding prevalent OXA carbapenemases in Acinetobacter spp. International Journal of Antimicrobial Agents [Internet]. 2006 Apr [cited 2024 Jun 22];27(4):351–3. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0924857906000264 pmid:16564159
- 24. Blaak H, Kemper MA, De Man H, Van Leuken JPG, Schijven JF, Van Passel MWJ, et al. Nationwide surveillance reveals frequent detection of carbapenemase-producing Enterobacterales in Dutch municipal wastewater. Science of The Total Environment [Internet]. 2021 Jul [cited 2024 Jun 22];776:145925. Available from: https://linkinghub.elsevier.com/retrieve/pii/S004896972100992X
- 25. Wang D, Berglund B, Li Q, Shangguan X, Li J, Liu F, et al. Transmission of clones of carbapenem-resistant Escherichia coli between a hospital and an urban wastewater treatment plant. Environmental Pollution [Internet]. 2023 Nov [cited 2024 Jun 22];336:122455. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0269749123014574 pmid:37633440
- 26. Too RJ, Gitao GC, Bebora LC, Mollenkopf DF, Kariuki SM, Wittum TE. Frequency and diversity of carbapenemase-producing Enterobacterales recovered from untreated wastewater impacted by selective media containing cefotaxime and meropenem in Ohio, USA. Aworh MK, editor. PLoS ONE [Internet]. 2023 Feb 22 [cited 2024 Jun 22];18(2):e0281909. Available from: https://dx.plos.org/10.1371/journal.pone.0281909 pmid:36812188
- 27. Lamba M, Graham DW, Ahammad SZ. Hospital Wastewater Releases of Carbapenem-Resistance Pathogens and Genes in Urban India. Environ Sci Technol [Internet]. 2017 Dec 5 [cited 2024 Jun 22];51(23):13906–12. Available from: https://pubs.acs.org/doi/10.1021/acs.est.7b03380 pmid:28949542
- 28. Azuma T, Uchiyama T, Zhang D, Usui M, Hayashi T. Distribution and characteristics of carbapenem-resistant and extended-spectrum β-lactamase (ESBL) producing Escherichia coli in hospital effluents, sewage treatment plants, and river water in an urban area of Japan. Science of The Total Environment [Internet]. 2022 Sep [cited 2024 Jun 22];839:156232. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0048969722033290
- 29. Bain R, Cronk R, Wright J, Yang H, Slaymaker T, Bartram J. Fecal Contamination of Drinking-Water in Low- and Middle-Income Countries: A Systematic Review and Meta-Analysis. Hunter PR, editor. PLoS Med [Internet]. 2014 May 6 [cited 2024 Jun 22];11(5):e1001644. Available from: https://dx.plos.org/10.1371/journal.pmed.1001644 pmid:24800926
- 30. Chelaru EC, Muntean AA, Hogea MO, Muntean MM, Popa MI, Popa GL. The Importance of Carbapenemase-Producing Enterobacterales in African Countries: Evolution and Current Burden. Antibiotics [Internet]. 2024 Mar 24 [cited 2024 Jun 22];13(4):295. Available from: https://www.mdpi.com/2079-6382/13/4/295 pmid:38666971
- 31. Revathi G, Siu LK, Lu PL, Huang LY. First report of NDM-1-producing Acinetobacter baumannii in East Africa. International Journal of Infectious Diseases [Internet]. 2013 Dec [cited 2024 Jun 22];17(12):e1255–8. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1201971213002609 pmid:24176550
- 32. Henson SP, Boinett CJ, Ellington MJ, Kagia N, Mwarumba S, Nyongesa S, et al. Molecular epidemiology of Klebsiella pneumoniae invasive infections over a decade at Kilifi County Hospital in Kenya. International Journal of Medical Microbiology [Internet]. 2017 Oct [cited 2024 Jun 22];307(7):422–9. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1438422117301121 pmid:28789913
- 33. Musila L, Kyany’a C, Maybank R, Stam J, Oundo V, Sang W. Detection of diverse carbapenem and multidrug resistance genes and high-risk strain types among carbapenem non-susceptible clinical isolates of target gram-negative bacteria in Kenya. Karunasagar I, editor. PLoS ONE [Internet]. 2021 Feb 22 [cited 2024 Jun 22];16(2):e0246937. Available from: https://dx.plos.org/10.1371/journal.pone.0246937 pmid:33617559
- 34. Ndiritu GG, Gichuki NN, Kaur P, Triest L. Characterization of environmental gradients using physico-chemical measurements and diatom densities in Nairobi River, Kenya. Aquatic Ecosystem Health and Management. 2003;6(3):343–54.
- 35. Haugland RA, Siefring SC, Wymer LJ, Brenner KP, Dufour AP. Comparison of Enterococcus measurements in freshwater at two recreational beaches by quantitative polymerase chain reaction and membrane filter culture analysis. Water Research [Internet]. 2005 Feb [cited 2024 Jun 22];39(4):559–68. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0043135404005342 pmid:15707628
- 36. Shrestha A, Dorevitch S. Evaluation of rapid qPCR method for quantification of E. coli at non-point source impacted Lake Michigan beaches. Water Research [Internet]. 2019 Jun [cited 2024 Jun 22];156:395–403. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0043135419302532
- 37. Nordmann P, Poirel L, Dortet L. Rapid Detection of Carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis. 2012 Sep;18(9):1503–7.
- 38. Bush K. A resurgence of β-lactamase inhibitor combinations effective against multidrug-resistant Gram-negative pathogens. International Journal of Antimicrobial Agents [Internet]. 2015 Nov [cited 2024 Jun 22];46(5):483–93. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0924857915003180
- 39. Österblad M, Hakanen AJ, Jalava J. Evaluation of the Carba NP Test for Carbapenemase Detection. Antimicrob Agents Chemother [Internet]. 2014 Dec [cited 2024 Jun 22];58(12):7553–6. Available from: https://journals.asm.org/doi/10.1128/AAC.02761-13 pmid:25246404
- 40. Dortet L, Poirel L, Nordmann P. Rapid Identification of Carbapenemase Types in Enterobacteriaceae and Pseudomonas spp. by Using a Biochemical Test. Antimicrob Agents Chemother. 2012 Dec;56(12):6437–40. pmid:23070158
- 41. Pasteran F, Tijet N, Melano RG, Corso A. Simplified Protocol for Carba NP Test for Enhanced Detection of Carbapenemase Producers Directly from Bacterial Cultures. Bourbeau P, editor. J Clin Microbiol [Internet]. 2015 Dec [cited 2024 Jun 22];53(12):3908–11. Available from: https://journals.asm.org/doi/10.1128/JCM.02032-15 pmid:26424841
- 42. Workneh M, Yee R, Simner PJ. Phenotypic Methods for Detection of Carbapenemase Production in Carbapenem-Resistant Organisms: What Method Should Your Laboratory Choose? Clinical Microbiology Newsletter [Internet]. 2019 Jan [cited 2024 Jun 22];41(2):11–22. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0196439919300017
- 43. Yigit H, Queenan AM, Anderson GJ, Domenech-Sanchez A, Biddle JW, Steward CD, et al. Novel Carbapenem-Hydrolyzing β-Lactamase, KPC-1, from a Carbapenem-Resistant Strain of Klebsiella pneumoniae. Antimicrob Agents Chemother [Internet]. 2001 Apr [cited 2024 Jun 22];45(4):1151–61. Available from: https://journals.asm.org/doi/10.1128/AAC.45.4.1151-1161.2001
- 44. Gröbner S, Linke D, Schütz W, Fladerer C, Madlung J, Autenrieth IB, et al. Emergence of carbapenem-non-susceptible extended-spectrum β-lactamase-producing Klebsiella pneumoniae isolates at the university hospital of Tübingen, Germany. Journal of Medical Microbiology [Internet]. 2009 Jul 1 [cited 2024 Jun 22];58(7):912–22. Available from: https://www.microbiologyresearch.org/content/journal/jmm/10.1099/jmm.0.005850-0
- 45. Peirano G, Pillai DR, Pitondo-Silva A, Richardson D, Pitout JDD. The characteristics of NDM-producing Klebsiella pneumoniae from Canada. Diagnostic Microbiology and Infectious Disease [Internet]. 2011 Oct [cited 2024 Jun 22];71(2):106–9. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0732889311002537 pmid:21924993
- 46. Mollenkopf DF, Mathys DA, Feicht SM, Stull JW, Bowman AS, Daniels JB, et al. Maintenance of Carbapenemase-Producing Enterobacteriaceae in a Farrow-to-Finish Swine Production System. Foodborne Pathogens and Disease [Internet]. 2018 Jun [cited 2024 Jun 22];15(6):372–6. Available from: http://www.liebertpub.com/doi/10.1089/fpd.2017.2355
- 47. Lob SH, Hackel MA, Kazmierczak KM, Young K, Motyl MR, Karlowsky JA, et al. In Vitro Activity of Imipenem-Relebactam against Gram-Negative ESKAPE Pathogens Isolated by Clinical Laboratories in the United States in 2015 (Results from the SMART Global Surveillance Program). Antimicrob Agents Chemother [Internet]. 2017 Jun [cited 2024 Jun 22];61(6):e02209–16. Available from: https://journals.asm.org/doi/10.1128/AAC.02209-16
- 48. Schechner V, Straus-Robinson K, Schwartz D, Pfeffer I, Tarabeia J, Moskovich R, et al. Evaluation of PCR-Based Testing for Surveillance of KPC-Producing Carbapenem-Resistant Members of the Enterobacteriaceae Family. J Clin Microbiol [Internet]. 2009 Oct [cited 2024 Aug 18];47(10):3261–5. Available from: https://journals.asm.org/doi/10.1128/JCM.02368-08
- 49. Ellington MJ, Kistler J, Livermore DM, Woodford N. Multiplex PCR for rapid detection of genes encoding acquired metallo- -lactamases. Journal of Antimicrobial Chemotherapy [Internet]. 2006 Nov 13 [cited 2024 Jun 22];59(2):321–2. Available from: https://academic.oup.com/jac/article-lookup/doi/10.1093/jac/dkl481
- 50. Hong SS, Kim K, Huh JY, Jung B, Kang MS, Hong SG. Multiplex PCR for rapid detection of genes encoding class A carbapenemases. Ann Lab Med. 2012 Sep;32(5):359–61. pmid:22950072
- 51. Dallenne C, Da Costa A, Decré D, Favier C, Arlet G. Development of a set of multiplex PCR assays for the detection of genes encoding important β-lactamases in Enterobacteriaceae. Journal of Antimicrobial Chemotherapy [Internet]. 2010 Mar [cited 2024 Aug 29];65(3):490–5. Available from: https://academic.oup.com/jac/article-lookup/doi/10.1093/jac/dkp498
- 52. Tijet N, Patel SN, Melano RG. Detection of carbapenemase activity in Enterobacteriaceae: comparison of the carbapenem inactivation method versus the Carba NP test: Table 1. J Antimicrob Chemother [Internet]. 2016 Jan [cited 2024 Jun 22];71(1):274–6. Available from: https://academic.oup.com/jac/article-lookup/doi/10.1093/jac/dkv283
- 53. Tacão M, Correia A, Henriques IS. Low Prevalence of Carbapenem-Resistant Bacteria in River Water: Resistance Is Mostly Related to Intrinsic Mechanisms. Microbial Drug Resistance [Internet]. 2015 Oct [cited 2024 Jun 22];21(5):497–506. Available from: http://www.liebertpub.com/doi/10.1089/mdr.2015.0072 pmid:26430939
- 54. Ebomah KE, Okoh AI. Detection of Carbapenem-Resistance Genes in Klebsiella Species Recovered from Selected Environmental Niches in the Eastern Cape Province, South Africa. Antibiotics [Internet]. 2020 Jul 21 [cited 2024 Jun 22];9(7):425. Available from: https://www.mdpi.com/2079-6382/9/7/425
- 55. Ruiz N, Silhavy TJ. How Escherichia coli Became the Flagship Bacterium of Molecular Biology. O’Toole G, editor. J Bacteriol [Internet]. 2022 Sep 20 [cited 2024 Jun 22];204(9):e00230–22. Available from: https://journals.asm.org/doi/10.1128/jb.00230-22
- 56. Poirel L, Aires-de-Sousa M, Kudyba P, Kieffer N, Nordmann P. Screening and Characterization of Multidrug-Resistant Gram-Negative Bacteria from a Remote African Area, São Tomé and Príncipe. Antimicrob Agents Chemother [Internet]. 2018 Sep [cited 2024 Jun 22];62(9):e01021–18. Available from: https://journals.asm.org/doi/10.1128/AAC.01021-18
- 57. Khan AU, Maryam L, Zarrilli R. Structure, Genetics and Worldwide Spread of New Delhi Metallo-β-lactamase (NDM): a threat to public health. BMC Microbiol [Internet]. 2017 Dec [cited 2024 Jun 22];17(1):101. Available from: http://bmcmicrobiol.biomedcentral.com/articles/10.1186/s12866-017-1012-8
- 58. Poirel L, Walsh TR, Cuvillier V, Nordmann P. Multiplex PCR for detection of acquired carbapenemase genes. Diagnostic Microbiology and Infectious Disease [Internet]. 2011 May [cited 2024 Jun 22];70(1):119–23. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0732889310005559 pmid:21398074
- 59. Villinger D, Schultze TG, Musyoki VM, Inwani I, Aluvaala J, Okutoyi L, et al. Genomic transmission analysis of multidrug-resistant Gram-negative bacteria within a newborn unit of a Kenyan tertiary hospital: A four-month prospective colonization study. Front Cell Infect Microbiol [Internet]. 2022 Aug 25 [cited 2024 Jun 22];12:892126. Available from: https://www.frontiersin.org/articles/10.3389/fcimb.2022.892126/full pmid:36093198
- 60. Edwards T, Williams CT, Olwala M, Andang’o P, Otieno W, Nalwa GN, et al. Molecular surveillance reveals widespread colonisation by carbapenemase and extended spectrum beta-lactamase producing organisms in neonatal units in Kenya and Nigeria. Antimicrob Resist Infect Control [Internet]. 2023 Feb 22 [cited 2024 Jun 22];12(1):14. Available from: https://aricjournal.biomedcentral.com/articles/10.1186/s13756-023-01216-0
- 61. Arnett H, Scordo K. Surveillance Screening to Reduce Carbapenem Resistance. The Journal for Nurse Practitioners [Internet]. 2019 Jun [cited 2024 Jun 22];15(6):434–7. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1555415518312984
- 62. ElMahallawy H, Zafer MM, Al-Agamy M, Amin MA, Mersal MM, Booq RY, et al. Dissemination of ST101 blaOXA-48 producing Klebsiella pneumoniae at tertiary care setting. J Infect Dev Ctries [Internet]. 2018 Jun 30 [cited 2024 Jun 22];12(06):422–8. Available from: https://jidc.org/index.php/journal/article/view/9789
- 63. Tafoukt R, Touati A, Leangapichart T, Bakour S, Rolain JM. Characterization of OXA-48-like-producing Enterobacteriaceae isolated from river water in Algeria. Water Research [Internet]. 2017 Sep [cited 2024 Jun 22];120:185–9. Available from: https://linkinghub.elsevier.com/retrieve/pii/S004313541730341X pmid:28486169
- 64. Ita T, Luvsansharav UO, Smith RM, Mugoh R, Ayodo C, Oduor B, et al. Prevalence of colonization with multidrug-resistant bacteria in communities and hospitals in Kenya. Sci Rep [Internet]. 2022 Dec 24 [cited 2024 Jun 22];12(1):22290. Available from: https://www.nature.com/articles/s41598-022-26842-3
- 65. Haeili M, Barmudeh S, Omrani M, Zeinalzadeh N, Kafil HS, Batignani V, et al. Whole-genome sequence analysis of clinically isolated carbapenem resistant Escherichia coli from Iran. BMC Microbiol [Internet]. 2023 Feb 27 [cited 2024 Jun 22];23(1):49. Available from: https://bmcmicrobiol.biomedcentral.com/articles/10.1186/s12866-023-02796-y pmid:36850019
- 66. Njuguna SM, Yan X, Gituru RW, Wang Q, Wang J. Assessment of macrophyte, heavy metal, and nutrient concentrations in the water of the Nairobi River, Kenya. Environ Monit Assess [Internet]. 2017 Sep [cited 2024 Jun 22];189(9):454. Available from: http://link.springer.com/10.1007/s10661-017-6159-0 pmid:28815343
- 67. Fayiga AO, Ipinmoroti MO, Chirenje T. Environmental pollution in Africa. Environ Dev Sustain [Internet]. 2018 Feb [cited 2024 Jun 22];20(1):41–73. Available from: http://link.springer.com/10.1007/s10668-016-9894-4
- 68. Mairi A, Pantel A, Sotto A, Lavigne JP, Touati A. OXA-48-like carbapenemases producing Enterobacteriaceae in different niches. Eur J Clin Microbiol Infect Dis [Internet]. 2018 Apr [cited 2024 Jun 22];37(4):587–604. Available from: http://link.springer.com/10.1007/s10096-017-3112-7 pmid:28990132
- 69. Ngumba E, Gachanja A, Tuhkanen T. Occurrence of selected antibiotics and antiretroviral drugs in Nairobi River Basin, Kenya. Science of The Total Environment [Internet]. 2016 Jan [cited 2024 Jun 22];539:206–13. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0048969715306410 pmid:26363393
- 70. Ngigi AN, Magu MM, Muendo BM. Occurrence of antibiotics residues in hospital wastewater, wastewater treatment plant, and in surface water in Nairobi County, Kenya. Environ Monit Assess [Internet]. 2020 Jan [cited 2024 Jun 22];192(1):18. Available from: http://link.springer.com/10.1007/s10661-019-7952-8
- 71. Kiyaga S, Kyany’a C, Muraya AW, Smith HJ, Mills EG, Kibet C, et al. Genetic Diversity, Distribution, and Genomic Characterization of Antibiotic Resistance and Virulence of Clinical Pseudomonas aeruginosa Strains in Kenya. Front Microbiol [Internet]. 2022 Mar 14 [cited 2024 Jun 22];13:835403. Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2022.835403/full
- 72. Guo J, Li J, Chen H, Bond PL, Yuan Z. Metagenomic analysis reveals wastewater treatment plants as hotspots of antibiotic resistance genes and mobile genetic elements. Water Research [Internet]. 2017 Oct [cited 2024 Jun 22];123:468–78. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0043135417305651 pmid:28689130
- 73. Mushi MF, Mshana SE, Imirzalioglu C, Bwanga F. Carbapenemase Genes among Multidrug Resistant Gram Negative Clinical Isolates from a Tertiary Hospital in Mwanza, Tanzania. BioMed Research International [Internet]. 2014 [cited 2024 Jun 22];2014:1–6. Available from: https://www.hindawi.com/journals/bmri/2014/303104/ pmid:24707481