Molecular epidemiology of multidrug-resistant Klebsiella pneumoniae, Enterobacter cloacae, and Escherichia coli outbreak among neonates in Tembisa hospital, South Africa.

Background: An outbreak of multidrug-resistant Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae infections in a neonatal ward within a tertiary hospital in South Africa resulted in the mortality of 10 patients within six months. In this work, the genomic epidemiology of and the molecular factors mediating this outbreak were investigated. Methods: Bacterial cultures obtained from clinical samples collected from the infected neonates underwent phenotypic and molecular analyses to determine their species, sensitivity to antibiotics, production of carbapenemases, complete resistance genes profile, clonality, epidemiology, and evolutionary relationships. Mobile genetic elements flanking the resistance genes and facilitating their spread were also characterized. Results: The outbreak was centered in two major wards and affected mainly neonates between September 2019 and March 2020. Most isolates (n = 27 isolates) were K. pneumoniae while both E. coli and E. cloacae had three isolates each. Notably, 33/34 isolates were multidrug resistant (MDR), with 30 being resistant to at least four drug classes. All the isolates were carbapenemase-positive, but four bla OXA-48 isolates were susceptible to carbapenems. Bla NDM-1 (n = 13) and bla OXA-48/181 (n = 15) were respectively found on IS91 and IS6-like IS26 composite transposons in the isolates alongside several other resistance genes. The repertoire of resistance and virulence genes, insertion sequences, and plasmid replicon types in the strains explains their virulence, resistance, and quick dissemination among the neonates. Conclusions: The outbreak of fatal MDR infections in the neonatal wards were mediated by clonal (vertical) and horizontal (plasmid-mediated) spread of resistant and virulent strains (and genes) that have been also circulating locally and globally.

Molecular Screening of Clinical Multidrug-Resistant Gram-Negative Bacteria Shows Endemicity of Carbapenemases, Coexistence of Multiple Carbapenemases, and Rarity of mcr in South Africa.

Background: Extensive use of carbapenems to treat multidrug-resistant (MDR) Gram-negative bacteria (GNB) facilitates the wide dissemination of carbapenemase-producing carbapenem-resistant GNB. Colistin was reintroduced into clinical settings to manage these GNB infections. However, there is currently an increase in the dissemination of mobile colistin resistance (mcr)-producing colistin-resistant GNB isolates in clinical settings. The epidemiology of carbapenemases and mcr in Pretoria was evaluated. Methods: Clinical MDR GNB were collected and screened for carbapenemases and mcr using polymerase chain reaction (PCR); their antibiotic susceptibility profiles were elucidated using the Vitek® 2 automated system (Biomerieux, France) and microbroth dilution (for colistin). Results and Discussion: A total of 306 isolates were collected; a majority of these were Klebsiella pneumoniae (n = 208) and were collected from males (n = 158). The isolates were retrieved from a variety of infection sites, including urine, blood cultures, and rectal swabs. The Vitek 2 system found that these isolates were largely resistant to ß-lactams, where 217 (70.9%) had reduced susceptibility to at least one carbapenem (ertapenem, meropenem, or imipenem), and 81 isolates (26.5%) were resistant to colistin. PCR screening identified 201 (65.7%) isolates harboring carbapenemase genes consisting of blaOXA-48 (170, 84.2%), blaNDM (31, 15.4%), blaIMP (5, 2%), blaKPC (4, 1%), and blaVIM (5, 2%). Furthermore, 14 blaOXA-48-producing isolates were coharboring blaVIM (2), blaNDM (9), blaKPC (1), and blaIMP (2) genes. Only one isolate harbored the mobile colistin resistance (mcr)-1 gene, and this is the first report of an mcr-1-producing Acinetobacter baumannii isolate in South Africa. Conclusion: There is high endemicity of carbapenemase genes and a low prevalence of mcr genes in GNB, particularly in K. pneumoniae, in health care facilities in Pretoria and surrounding regions of South Africa. Significance: Health care facilities in Pretoria are becoming breeding grounds for MDR infections that threaten public health. Careful use of carbapenems and other antibiotics is necessary to prevent further escalation and outbreak of these MDR strains that can claim several lives.

Global epidemiology, genetic environment, risk factors and therapeutic prospects of mcr genes: A current and emerging update.

Background: Mobile colistin resistance (mcr) genes modify Lipid A molecules of the lipopolysaccharide, changing the overall charge of the outer membrane. Results and discussion: Ten mcr genes have been described to date within eleven Enterobacteriaceae species, with Escherichia coli, Klebsiella pneumoniae, and Salmonella species being the most predominant. They are present worldwide in 72 countries, with animal specimens currently having the highest incidence, due to the use of colistin in poultry for promoting growth and treating intestinal infections. The wide dissemination of mcr from food animals to meat, manure, the environment, and wastewater samples has increased the risk of transmission to humans via foodborne and vector-borne routes. The stability and spread of mcr genes were mediated by mobile genetic elements such as the IncHI2 conjugative plasmid, which is associated with multiple mcr genes and other antibiotic resistance genes. The cost of acquiring mcr is reduced by compensatory adaptation mechanisms. MCR proteins are well conserved structurally and via enzymatic action. Thus, therapeutics found effective against MCR-1 should be tested against the remaining MCR proteins. Conclusion: The dissemination of mcr genes into the clinical setting, is threatening public health by limiting therapeutics options available. Combination therapies are a promising option for managing and treating colistin-resistant Enterobacteriaceae infections whilst reducing the toxic effects of colistin.

Current and emerging polymyxin resistance diagnostics: A systematic review of established and novel detection methods.

The emergence of polymyxin resistance, due to transferable mcr genes, threatens public and animal health as there are limited therapeutic options. As polymyxin is one of the last-line antibiotics, there is a need to contain the spread of its resistance to conserve its efficacy. Herein, we describe current and emerging polymyxin resistance diagnostics to inform faster clinical diagnostic choices. A literature search in diverse databases for studies published between 2016 and 2020 was performed. English articles evaluating colistin resistance methods/diagnostics were included. Screening resulted in the inclusion of 93 journal articles. Current colistin resistance diagnostics are either phenotypic or molecular. Broth microdilution is currently the only gold standard for determining colistin MICs (minimum inhibitory concentration). Phenotypic methods comprise of agar-based methods such as CHROMagar™ Col-APSE, SuperPolymyxin, ChromID® Colistin R, LBJMR and LB medium; manual MIC-determiners viz., UMIC, MICRONAUT MIC-Strip and ComASP Colistin; automated antimicrobial susceptibility testing systems such as BD Phoenix, MICRONAUT-S, MicroScan, Sensititre and Vitek 2; MCR-detectors such as lateral flow immunoassay (LFI) and chelator-based assays including EDTA- and DPA-based tests, that is, combined disk test, modified colistin broth-disk elution (CBDE), Colispot, and Colistin MAC test as well as biochemical colorimetric tests, that is, Rapid Polymyxin NP test and Rapid ResaPolymyxin NP test. Molecular methods only characterize mobile colistin resistance; they include PCR, LAMP and whole-genome sequencing. Due to the faster turnaround time (≤3 h), improved sensitivity (84%-100%) and specificity (93.3%-100%) of the Rapid ResaPolymyxin NP test and Fastinov® , we recommend this test for initial screening of colistin-resistant isolates. This can be followed by CBDE with EDTA or the LFI as they both have 100% sensitivity and a specificity of ≥94.3% for the rapid screening of mcr genes. However, molecular assays such as LAMP and PCR may be considered in well-equipped clinical laboratories.

Emerging Transcriptional and Genomic Mechanisms Mediating Carbapenem and Polymyxin Resistance in Enterobacteriaceae: a Systematic Review of Current Reports.

The spread of carbapenem- and polymyxin-resistant Enterobacteriaceae poses a significant threat to public health, challenging clinicians worldwide with limited therapeutic options. This review describes the current coding and noncoding genetic and transcriptional mechanisms mediating carbapenem and polymyxin resistance, respectively. A systematic review of all studies published in PubMed database between 2015 to October 2020 was performed. Journal articles evaluating carbapenem and polymyxin resistance mechanisms, respectively, were included. The search identified 171 journal articles for inclusion. Different New Delhi metallo-ß-lactamase (NDM) carbapenemase variants had different transcriptional and affinity responses to different carbapenems. Mutations within the Klebsiella pneumoniae carbapenemase (KPC) mobile transposon, Tn4401, affect its promoter activity and expression levels, increasing carbapenem resistance. Insertion of IS26 in ardK increased imipenemase expression 53-fold. ompCF porin downregulation (mediated by envZ and ompR mutations), micCF small RNA hyperexpression, efflux upregulation (mediated by acrA, acrR, araC, marA, soxS, ramA, etc.), and mutations in acrAB-tolC mediated clinical carbapenem resistance when coupled with ß-lactamase activity in a species-specific manner but not when acting without ß-lactamases. Mutations in pmrAB, phoPQ, crrAB, and mgrB affect phosphorylation of lipid A of the lipopolysaccharide through the pmrHFIJKLM (arnBCDATEF or pbgP) cluster, leading to polymyxin resistance; mgrB inactivation also affected capsule structure. Mobile and induced mcr, efflux hyperexpression and porin downregulation, and Ecr transmembrane protein also conferred polymyxin resistance and heteroresistance. Carbapenem and polymyxin resistance is thus mediated by a diverse range of genetic and transcriptional mechanisms that are easily activated in an inducing environment. The molecular understanding of these emerging mechanisms can aid in developing new therapeutics for multidrug-resistant Enterobacteriaceae isolates.

Emergence of mcr-9.1 in Extended-Spectrum-ß-Lactamase-Producing Clinical Enterobacteriaceae in Pretoria, South Africa: Global Evolutionary Phylogenomics, Resistome, and Mobilome.

Extended-spectrum-ß-lactamase (ESBL)-producing Enterobacteriaceae are critical-priority pathogens that cause substantial fatalities. With the emergence of mobile mcr genes mediating resistance to colistin in Enterobacteriaceae, clinicians are now left with few therapeutic options. Eleven clinical Enterobacteriaceae strains with resistance to cephems and/or colistin were genomically analyzed to determine their resistomes, mobilomes, and evolutionary relationships to global strains. The global phylogenomics of mcr genes and mcr-9.1-bearing genomes were further analyzed. Ten isolates were ESBL positive. The isolates were multidrug resistant and phylogenetically related to global clones but distant from local strains. Multiple resistance genes, including bla CTX-M-15 bla TEM-1, and mcr-9.1, were found in single isolates; ISEc9, IS19, and Tn3 transposons bracketed bla CTX-M-15 and bla TEM-1 Common plasmid types included IncF, IncH, and ColRNAI. mcr-9 was of close sequence identity to mcr-3, mcr-5, mcr-7, mcr-8, and mcr-10. Genomes bearing mcr-9.1 clustered into six main phyletic groups (A to F), with those of this study belonging to clade B. Enterobacter species and Salmonella species are the main hosts of mcr-9.1 globally, although diverse promiscuous plasmids disseminate mcr-9.1 across different bacterial species. Emergence of mcr-9.1 in ESBL-producing Enterobacteriaceae in South Africa is worrying, due to the restricted therapeutic options. Intensive One Health molecular surveillance might discover other mcr alleles and inform infection management and antibiotic choices.IMPORTANCE Colistin is currently the last-resort antibiotic for difficult-to-treat bacterial infections. However, colistin resistance genes that can move from bacteria to bacteria have emerged, threatening the safe treatment of many bacterial infections. One of these genes, mcr-9.1, has emerged in South Africa in bacteria that are multidrug resistant, further limiting treatment options for clinicians. In this work, we show that this new gene is disseminating worldwide through Enterobacter and Salmonella species through multiple plasmids. This worrying observation requires urgent action to prevent further escalation of this gene in South Africa and Africa.

Comparative Evaluation of CHROMagar COL-APSE, MicroScan Walkaway, ComASP Colistin, and Colistin MAC Test in Detecting Colistin-resistant Gram-Negative Bacteria.

Colistin has become a critical antibiotic for fatal Gram-negative infections owing to the proliferation of multidrug-resistant carbapenemase-producing bacteria. Thus, cheaper, faster, efficient and easier-to-use colistin diagnostics are required for clinical surveillance, diagnoses and therapeutics. The sensitivity, specificity, major error (ME), very major error (VME), categorial agreement, essential agreement, turnaround time (TAT), average cost, and required skill for four colistin resistance diagnostics viz., CHROMagar COL-APSE, ComASP Colistin, MicroScan, and Colistin MAC Test (CMT) were evaluated against broth microdilution (BMD) using 84 Gram-negative bacterial isolates. A multiplex PCR (M-PCR) was used to screen all isolates to detect the presence of the mcr-1 to mcr-5 genes. A 15-point grading scale was used to grade the tests under skill, ease, processing time etc. mcr-1 was detected by both M-PCR and CMT in a single E. coli isolate, with other PCR amplicons suggestive of mcr-2, -3 and -4 genes being also observed on the gel. The sensitivity and specificity of CHROMagar COL-APSE, MicroScan, and ComASP Colistin, were 82.05% and 66.67%, 92.31% and 76.92%, and 100% and 88.89% respectively. The MicroScan was the most expensive at a cost (per sampe tested) of R221.6 ($15.0), followed by CHROMagar COL-APSE (R118.3; $8.0), M-PCR (R75.1; $5.1), CMT (R20.1; $1.4) and ComASP Colistin (R2.64; $0.2). CHROMagar was the easiest to perform, followed by ComASP Colistin, M-PCR, MicroScan, CMT and BMD whilst M-PCR and MicroScan required higher skill. The ComASP Colistin was the best performing diagnostic test, with low VME and ME, making it recommendable for routine colistin sensitivity testing in clinical laboratories; particularly, in poorer settings. It is however limited by a TAT of 18-24 hours.

Pathogenomics and Evolutionary Epidemiology of Multi-Drug Resistant Clinical Klebsiella pneumoniae Isolated from Pretoria, South Africa.

Antibiotic-resistant Klebsiella pneumoniae is increasingly being implicated in invasive infections worldwide with high mortalities. Forty-two multidrug resistant (MDR) K. pneumoniae isolates were collected over a 4-month period. Antimicrobial susceptibility was determined using Microscan. The evolutionary epidemiology, resistome, virulome and mobilome of the isolates were characterised using whole-genome sequencing and bioinformatics analysis. All isolates contained the blaCTX-M gene, whilst 41/42(97%) contained blaTEM, 36/42(86%) contained blaOXA and 35/42(83%) harboured blaSHV genes. Other resistance genes found included blaLEN, aac(6')-lb-cr, qnrA, qnrB, qnrS, oqxAB, aad, aph, dfr, sul1, sul2, fosA, and cat genes. Fluoroquinolone and colistin resistance-conferring mutations in parC, gyrAB, pmrAB, phoPQ and kpnEF were identified. The blaLEN gene, rarely described worldwide, was identified in four isolates. The isolates comprised diverse sequence types, the most common being ST152 in 7/42(17%) isolates; clone-specific O and K capsule types were identified. Diverse virulence genes that were not clone-specific were identified in all but one isolate. IncF, IncH and IncI plasmid replicons and two novel integrons were present. The blaCTX-M-15 and blaTEM-1 genes were bracketed by Tn3 transposons, ISEc9, a resolvase and IS91 insertion sequence. There were 20 gene cassettes in 14 different cassette arrays, with the dfrA and aadA gene cassettes being the most frequent. Phylogenetic analysis demonstrated that the isolates were evolutionarily associated with strains from both South Africa and abroad. These findings depict the rich resistome, mobilome and virulome repertoire in clinical K. pneumoniae strains, which are mainly transmitted by clonal, multiclonal and horizontal means in South Africa.

Publisher Correction: The Resistome, Mobilome, Virulome and Phylogenomics of Multidrug-Resistant Escherichia coli Clinical Isolates from Pretoria, South Africa.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genomic analysis of a multidrug-resistant clinical Providencia rettgeri (PR002) strain with the novel integron ln1483 and an A/C plasmid replicon.

Whole-genome sequence analysis was performed on a multidrug-resistant Providencia rettgeri PR002 clinical strain isolated from the urine of a hospitalized patient in Pretoria, South Africa, in 2013. The resistome, mobilome, pathogenicity island(s), as well as virulence and heavy-metal resistance genes of the isolate, were characterized using whole-genome sequencing and bioinformatic analysis. PR002 had a genome assembly size of 4,832,624 bp with a GC content of 40.7%, an A/C2 plasmid replicase gene, four integrons/gene cassettes, 17 resistance genes, and several virulence and heavy metal resistance genes, confirming PR002 as a human pathogen. A novel integron, In1483, harboring the gene blaOXA-2 , was identified, with other uncharacterized class 1 integrons harboring aacA4cr and dfrA1. Aac(3')-IIa and blaSCO-1 , as well as blaPER-7 , sul2, and tet(B), were found bracketed by composite Tn3 transposons, and IS91, IS91, and IS4 family insertion sequences, respectively. PR002 was resistant to all antibiotics tested except amikacin, carbapenems, cefotaxime-clavulanate, ceftazidime-clavulanate, cefoxitin, and fosfomycin. PR002 was closely related to PR1 (USA), PRET_2032 (SPAIN), DSM_1131, and NCTC7477 clinical P. rettgeri strains, but not close enough to suggest it was imported into South Africa from other countries. Multidrug resistance in P. rettgeri is rare, particularly in clinical settings, making this case an important incident requiring urgent attention. This is also the first report of an A/C plasmid in P. rettgeri. The array, multiplicity, and diversity of resistance and virulence genes in this strain are concerning, necessitating stringent infection control, antibiotic stewardship, and periodic resistance surveillance/monitoring policies to preempt further horizontal and vertical spread of these resistance genes.

The Resistome, Mobilome, Virulome and Phylogenomics of Multidrug-Resistant Escherichia coli Clinical Isolates from Pretoria, South Africa.

Antibiotic-resistant Escherichia coli is a common occurrence in food, clinical, community and environmental settings worldwide. The resistome, mobilome, virulome and phylogenomics of 20 multidrug resistant (MDR) clinical E. coli isolates collected in 2013 from Pretoria, South Africa, were characterised. The isolates were all extended-spectrum ß-lactamase producers, harbouring CTX-M (n = 16; 80%), TEM-1B (n = 10; 50%) and OXA (n = 12, 60%) ß-lactamases alongside genes mediating resistance to fluoroquinolones, aminoglycosides, tetracyclines etc. Most resistance determinants were found on contigs containing IncF plasmid replicons and bracketed by composite transposons (Tn3), diverse ISs and class 1 integrons (In13, In54, In369, and In467). Gene cassettes such as blaOXA, dfrA5-psp-aadA2-cmlA1a-aadA1-qac and estX3-psp-aadA2-cmlA1a-aadA1a-qac were encompassed by Tn3 and ISs; several isolates had same or highly similar genomic antibiotic resistance islands. ST131 (n = 10), ST617 (n = 2) and singletons of ST10, ST73, ST95, ST410, ST648, ST665, ST744 and ST998 clones were phylogenetically related to clinical (human and animal) strains from Egypt, Kenya, Niger, Nigeria, Tanzania, and UK. A rich repertoire of virulence genes, including iss, gad and iha were identified. MDR E. coli harbouring chromosomal and plasmid-borne resistance genes in same and multiple clones exist in South Africa, which is very worrying for clinical epidemiology and infectious diseases management.

Phylogenomic and epidemiological insights into two clinical Mycobacterium bovis BCG strains circulating in South Africa.

BACKGROUND: Mycobacterium bovis BCG is a live, attenuated tuberculosis vaccine. While the vaccine protects infants from tuberculosis, complications including disseminated infections have been reported following vaccination. Genetically diverse BCG sub-strains now exist following continuous passaging of the original Pasteur strain for vaccine manufacture. This genetic diversity reportedly influences the severity of disseminated BCG infections and the efficacy of BCG immunization. METHODS: M. bovis BCG was isolated from infants suspected of being infected with tuberculosis. The whole genome of the clinical isolates and BCG Moscow were sequenced using Illumina Miseq and the sequences were analysed using CLC Genomics Workbench 7.0, PhyResSE v1.0, and Parsnp. RESULTS AND CONCLUSIONS: Genetic variations between the clinical strains and the reference BCG Copenhagen were identified. The clinical strains shared only one mutation in a secretion protein. Mutations were identified in various antibiotic resistance genes in the BCG isolates, which suggests their potential as multidrug-resistant (MDR) phenotypes. Phylogenetic analysis showed that the two isolates were distantly related, and the M1_S48 clinical isolate was closely related to M. bovis BCG Moscow. The phylogenomics results imply that two different BCG strains may be circulating in South Africa. However, it is difficult to associate the BCG vaccine strain administered and the BCG strain supplied with specific adverse events, as BCGiosis is under-reported. This study presents background genomic information for future surveillance and tracking of the distribution of BCGiosis-associated mycobacteria. It is also the first to report on the genomes of clinical BCG strains in Africa.

Plasmid evolution in carbapenemase-producing Enterobacteriaceae: a review.

Carbapenem-resistant Enterobacteriaceae (CRE) have been listed by the WHO as high-priority pathogens owing to their high association with mortalities and morbidities. Resistance to multiple ß-lactams complicates effective clinical management of CRE infections. Using plasmid typing methods, a wide distribution of plasmid replicon groups has been reported in CREs around the world, including IncF, N, X, A/C, L/M, R, P, H, I, and W. We performed a literature search for English research papers, published between 2013 and 2018, reporting on plasmid-mediated carbapenem resistance. A rise in both carbapenemase types and associated plasmid replicon groups was seen, with China, Canada, and the United States recording a higher increase than other countries. blaKPC was the most prevalent, except in Angola and the Czech Republic, where OXA-181 (n = 50, 88%) and OXA-48-like (n = 24, 44%) carbapenemases were most prevalent, respectively; blaKPC-2/3 accounted for 70% (n = 956) of all reported carbapenemases. IncF plasmids were found to be responsible for disseminating different antibiotic resistance genes worldwide, accounting for almost 40% (n = 254) of plasmid-borne carbapenemases. blaCTX-M , blaTEM , blaSHV , blaOXA-1/9 , qnr, and aac-(6')-lb were mostly detected concurrently with carbapenemases. Most reported plasmids were conjugative but not present in multiple countries or species, suggesting limited interspecies and interboundary transmission of a common plasmid. A major limitation to effective characterization of plasmid evolution was the use of PCR-based instead of whole-plasmid sequencing-based plasmid typing.

Draft Genome Sequence of a Clinical Enterococcus faecium Sequence Type 18 Strain from South Africa.

We report the first draft genome sequence of an Enterococcus faecium sequence type 18 (ST18) strain isolated from a tuberculosis patient in Africa. The genome is comprised of 3,202,539 bp, 501 contigs, 37.70% GC content, 3,202 protein-encoding genes, and 61 RNA genes. The resistome and virulome of this important pathogen are presented herein.

First Report of a Whole-Genome Shotgun Sequence of a Clinical Enterococcus faecalis Sequence Type 6 Strain from South Africa.

Enterococcus faecalis is a lactic acid-producing Gram-positive bacterium commonly found in the intestinal tract of humans and animals; it is implicated in multidrug-resistant nosocomial infections. The draft genome of this E. faecalis sequence type 6 (ST6) strain consists of 3,215,228 bp, with 37.20% GC content, 3,048 predicted coding sequences, and 61 RNA genes.