Mushal Allam

Genome Sequencing of Extended-Spectrum β-Lactamase (ESBL)-Producing Klebsiella pneumoniae Isolated from Pigs and Abattoir Workers in Cameroon

Background and objectives: Extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae is a serious public health issue globally. In this study, the antibiotic resistance genes, virulence factors, mobile genetic elements, and genetic lineages of circulating ESBL-producing K. pneumoniae strains isolated from pigs and humans in Cameroonian abattoirs were investigated using whole genome sequencing (WGS), in order to ascertain zoonotic transmission (viz. from animals to humans and/or vice-versa) in the food chain. Methods: During March–October 2016, 288 nasal and rectal pooled samples from 432 pigs as well as nasal and hand swabs from 82 humans were collected from Cameroon and South Africa. Seven ESBL-producing K. pneumoniae circulating in Cameroonian pig abattoirs were selected and their genomic DNA sequenced using an Illumina MiSeq platform. Generated reads were de novo assembled using the Qiagen CLC Genomics Workbench and SPAdes. The assembled contigs were annotated using RAST and antibiotic resistance genes, virulence factors, plasmids, and bacteriophages were identified with ResFinder, Virulence Finder, PlasmidFinder, and PHAST, respectively. Results: ESBL-producing K. pneumoniae were detected in pigs (34/158; 21.52%) and exposed workers (8/71; 11.26%) in Cameroon only. The circulating K. pneumoniae strains were dominated principally by the sequence type (ST) 14 and 39. In addition, the “high-risk” ST307 clone and two novel STs assigned ST2958 and ST2959 were detected. Genomic analysis identified various antibiotic resistance genes associated with resistance to β-lactams, aminoglycosides, fluoroquinolones, macrolide, lincosamide and streptogramins, rifampicin, sulfonamides, trimethoprim, phenicols and tetracycline. None of the ESBL-producing K. pneumoniae harbored virulence genes. Intermingled K. pneumoniae populations were observed between pig- and human-source within and across abattoirs in the country. Conclusion: Our study shows that ESBL-producing K. pneumoniae is actively disseminating in pigs and occupationally exposed workers in Cameroonian pig abattoirs and is probably underestimated in the absence of molecular epidemiological studies. It suggests pigs, abattoir workers and food products as potential reservoirs and sources of zoonotic transmission in Cameroon. Our findings underline the existence of a potential unheeded food safety and public health threat associated with these resistant strains and reinforce the crucial importance of implementing appropriate food safety measures and promoting rational antibiotic use.

Molecular characterization of invasive capsule null Neisseria meningitidis in South Africa

BackgroundThe meningococcal capsule is an important virulence determinant. Unencapsulated meningococci lacking capsule biosynthesis genes and containing the capsule null locus (cnl) are predominantly non-pathogenic. Rare cases of invasive meningococcal disease caused by cnl isolates belonging to sequence types (ST) and clonal complexes (cc) ST-845 (cc845), ST-198 (cc198), ST-192 (cc192) and ST-53 (cc53) have been documented. The clinical significance of these isolates however remains unclear. We identified four invasive cnl meningococci through laboratory-based surveillance in South Africa from 2003 through 2013, which we aimed to characterize using whole genome data.ResultsOne isolate [NG: P1.7-2,30: F1-2: ST-53 (cc53)] contained cnl allele 12, and caused empyema in an adult male with bronchiectasis from tuberculosis, diabetes mellitus and a smoking history. Three isolates were NG: P1.18-11,42-2: FΔ: ST-192 (cc192) and contained cnl allele 2. One patient was an adolescent male with meningitis. The remaining two isolates were from recurrent disease episodes (8 months apart) in a male child with deficiency of the sixth complement component, and with the exception of two single nucleotide polymorphisms, contained identical core genomes. The ST-53 (cc53) isolate possessed alleles for NHBA peptide 191 and fHbp variant 2; whilst the ST-192 (cc192) isolates contained NHBA peptide 704 and fHbp variant 3. All four isolates lacked nadA. Comparison of the South African genomes to 61 additional cnl genomes on the PubMLST Neisseria database (http://pubmlst.org/neisseria/), determined that most putative virulence genes could be found in both invasive and carriage phenotypes.ConclusionsAlthough rare, invasive disease by cnl meningococci may be associated with host immunodeficiency and such patients may benefit from protein-based meningococcal vaccines.

A novel adenovirus isolated from the Egyptian fruit bat in South Africa is closely related to recent isolates from China

Recently a number of novel adenoviruses have been isolated from diverse bat species and from diverse geographical locations. We describe the isolation of a novel adenovirus (Family Adenoviridae, genus Mastadenovirus) from a pool of liver and spleen tissue of an apparently healthy wild-caught Egyptian fruit bat (Rousettus aegyptiacus) in South Africa. Genetically the virus is most closely related to four mastadenoviruses recently isolated in China, from Miniopterus schreibersi and Rousettus leschenaultii bats, which are highly divergent from previously identified bat adenoviruses. The length of the Rousettus aegyptiacus adenovirus-3085 (RaegAdV-3085) genome, at 29,342 bp is similar to its closest relatives, and contains 27 open reading frames. The RaegAdV-3085 genome has a low G + C content (36.4%) relative to other viruses in the genus (between 43.6 and 63.9%) but similar to its closest relatives. The inverted terminal repeat (ITR) of RaegAdV-3085 is only 40 bp compared to between 61 and 178 bp of its closest relatives. The discovery of RaegAdV-3085 expands the diversity of known adenoviruses in bats and might represent a member of a new mastadenovirus species in bats.

Molecular Characterization of Corynebacterium diphtheriae Outbreak Isolates, South Africa, March–June 2015

In 2015, a cluster of respiratory diphtheria cases was reported from KwaZulu-Natal Province in South Africa. By using whole-genome analysis, we characterized 21 Corynebacterium diphtheriae isolates collected from 20 patients and contacts during the outbreak (1 patient was infected with 2 variants of C. diphtheriae). In addition, we included 1 cutaneous isolate, 2 endocarditis isolates, and 2 archived clinical isolates (ca. 1980) for comparison. Two novel lineages were identified, namely, toxigenic sequence type (ST) ST-378 (n = 17) and nontoxigenic ST-395 (n = 3). One archived isolate and the cutaneous isolate were ST-395, suggesting ongoing circulation of this lineage for >30 years. The absence of preexisting molecular sequence data limits drawing conclusions pertaining to the origin of these strains; however, these findings provide baseline genotypic data for future cases and outbreaks. Neither ST has been reported in any other country; this ST appears to be endemic only in South Africa.

Identification and characterization of microRNAs expressed in the African malaria vector Anopheles funestus life stages using high throughput sequencing

BackgroundOver the past several years, thousands of microRNAs (miRNAs) have been identified in the genomes of various insects through cloning and sequencing or even by computational prediction. However, the number of miRNAs identified in anopheline species is low and little is known about their role. The mosquito Anopheles funestus is one of the dominant malaria vectors in Africa, which infects and kills millions of people every year. Therefore, small RNA molecules isolated from the four life stages (eggs, larvae, pupae and unfed adult females) of An. funestus were sequenced using next generation sequencing technology.ResultsHigh throughput sequencing of four replicates in combination with computational analysis identified 107 mature miRNA sequences expressed in the An. funestus mosquito. These include 20 novel miRNAs without sequence identity in any organism and eight miRNAs not previously reported in the Anopheles genus but are known in non-anopheles mosquitoes. Finally, the changes in the expression of miRNAs during the mosquito development were determined and the analysis showed that many miRNAs have stage-specific expression, and are co-transcribed and co-regulated during development.ConclusionsThis study presents the first direct experimental evidence of miRNAs in An. funestus and the first profiling study of miRNA associated with the maturation in this mosquito. Overall, the results indicate that miRNAs play important roles during the growth and development. Silencing such molecules in a specific life stage could decrease the vector population and therefore interrupt malaria transmission.

Biological crusts of serpentine and non-serpentine soils from the Barberton Greenstone Belt of South Africa

Climate and geography can influence biological soil crust (BSC) community composition, but local heterogeneity in variables such as soil characteristics or microclimate gradients can also impact cryptogamic diversity. Heavy metals and nutrient imbalances in serpentine soils are known to influence the distributions of higher plants, but cryptogamic species appear to be more tolerant of substrate. The aim of this study was to compare the cryptogamic composition of serpentine and non-serpentine soils by using integrative taxonomy, which combines morphological and DNA barcoding data, to determine how soil characteristics in combination with rainfall can influence BSC community composition. Samples from serpentine and non-serpentine soils were enumerated and total genomic DNA was isolated from the soil samples. Analyses of the 16S rRNA gene and ITS sequences were done using the quantitative insights into microbial ecology (QIIME) workflow to determine which eukaryotic microorganisms were present in the samples. Sixty genera from the Cyanophyceae (38), Chlorophyceae (10), Bacillariophyceae (6), Eustigmatophyceae (4), Trebouxiophyceae (1) and Xanthophyceae (1) classes were detected with this approach. Results confirm that algae and cyanobacteria are tolerant of most substrates and can even colonize environments with high levels of heavy metal and nutrient imbalances, if moisture is present. Genera such as Acaryochloris, Annamia, Brasilonema, Chrocosphaera, Halomicronema, Planktothricoides, Rubidibacter, and Toxopsis are reported for the first time for South African soil.

Phylogenetic Analysis of Invasive Serotype 1 Pneumococcus in South Africa, 1989 to 2013

ABSTRACT Serotype 1 is an important cause of invasive pneumococcal disease in South Africa and has declined following the introduction of the 13-valent pneumococcal conjugate vaccine in 2011. We genetically characterized 912 invasive serotype 1 isolates from 1989 to 2013. Simpsons diversity index (D) and recombination ratios were calculated. Factors associated with sequence types (STs) were assessed. Clonal complex 217 represented 96% (872/912) of the sampled isolates. Following the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13), ST diversity increased in children <5 years (D, 0.39 to 0.63, P = 0.002) and individuals >14 years (D, 0.35 to 0.54, P < 0.001): ST-217 declined proportionately in children <5 years (153/203 [75%] versus 21/37 [57%], P = 0.027) and individuals >14 years (242/305 [79%] versus 96/148 [65%], P = 0.001), whereas ST-9067 increased (4/684 [0.6%] versus 24/228 [11%], P < 0.001). Three subclades were identified within ST-217: ST-217C1 (353/382 [92%]), ST-217C2 (15/382 [4%]), and ST-217C3 (14/382 [4%]). ST-217C2, ST-217C3, and single-locus variant (SLV) ST-8314 (20/912 [2%]) were associated with nonsusceptibility to chloramphenicol, tetracycline, and co-trimoxazole. ST-8314 (20/912 [2%]) was also associated with increased nonsusceptibility to penicillin (P < 0.001). ST-217C3 and newly reported ST-9067 had higher recombination ratios than those of ST-217C1 (4.344 versus 0.091, P < 0.001; and 0.086 versus 0.013, P < 0.001, respectively). Increases in genetic diversity were noted post-PCV13, and lineages associated with antimicrobial nonsusceptibility were identified.

Two cases of serotypeable and non-serotypeable variants of Streptococcus pneumoniae detected simultaneously during invasive disease

BackgroundMore than 94 serotypes of Streptococcus pneumoniae have been described to date, however the majority of disease is caused by approximately 20 serotypes. Some pneumococci do not react with commercially available antisera used for serotyping and are thus regarded as non-serotypeable (NT). These pneumococci are commonly isolated during carriage studies and very rarely cause invasive disease. Colonization may occur with more than one serotype however disease with more than one serotype is rarely detected. Thus there are limited data describing cases of pneumococcal disease caused by more than one isolate.ResultsIn two cases of invasive pneumococcal disease in South Africa, a non-serotypeable and a serotypeable isolate were co-detected during routine serotyping. A serotype 1 and 18C isolate were each co-detected with a non-serotypeable isolate in 2009 (case A) and 2010 (case B), from cerebrospinal fluid and blood, respectively. Both patients were 10–14 years old. For case A, the serotypeable isolate could not be obtained due to low representation in the mixed culture. Using electron microscopy we confirmed lack of capsule for the non-serotypeable isolates. Comparison of the case A non-serotypeable isolate with a serotype 1 genome revealed only the presence of the rhamnose biosynthesis genes (rmlA, B, C and D) in the capsular locus, all other capsular genes were absent. Nonetheless it had a multilocus sequence type (ST) associated with serotype 1 (ST217 and ribosomal ST3462) and its core genome clustered with other ST217 isolates. The case B non-serotypeable isolate had all serotype 18C capsular genes except for variation in the wchA and wze genes, compared to the 18C isolate. Both case B isolates were ST9817 and their core genomes were identical.ConclusionsThe ability of pneumococci to alter capsule production is a potential vaccine escape mechanism and therefore non-serotypeable pneumococci should be monitored as such organisms may increase under vaccine pressure.

Draft genome sequences of extended-spectrum β-lactamase-producing Enterobacter aerogenes isolated from swine and human

OBJECTIVES The draft genome sequences of two Enterobacter aerogenes strains (HN503E2II and PN108E5IIB) isolated from two Cameroonian abattoirs are reported. METHODS Bacterial genomic DNA of the two isolates was sequenced using an Illumina MiSeq platform. Generated reads were de novo assembled using CLC Genomics Workbench and SPAdes. The assembled contigs were annotated, and antibiotic resistance genes, virulence factors and plasmids were identified. RESULTS Whole-genome sequencing revealed that both strains were similar, with genomes of 4878638bp and 4794257bp, encoding several resistance genes associated with resistance to β-lactams, fluoroquinolones, aminoglycosides, fosfomycin, phenicols, sulphonamides, trimethoprim, macrolides and tetracycline. In silico analysis also revealed chromosomal integration of one plasmid in the genome of PN108E5IIB. CONCLUSION The genome sequences reported here will provide useful information for a better understanding of the genetic structure of E. aerogenes in Africa.

Draft Genome Sequence of Methicillin Resistant Staphylococcus epidermidis Isolate from Swine

OBJECTIVES Here we report the draft genome sequence of a methicillin-resistant Staphylococcus epidermidis strain (sequence type 59) isolated from a pooled rectal sample from pigs collected in an abattoir in South Africa. METHODS Genomic DNA of S. epidermidis PR246B0 was sequenced using an Illumina MiSeq platform. Generated reads were de novo assembled using CLC Genomics Workbench (QIAGEN). The assembled contigs were annotated and antimicrobial resistance genes, plasmids and the sequence type were identified. RESULTS The genome comprised a circular chromosome of 2537769bp, with a G-C content of 32.32% and various antimicrobial resistance genes associated with resistance to β-lactams, fluoroquinolones, aminoglycosides, fosfomycin, macrolides, lincosamides and tetracycline. Genome analysis also revealed the presence of seven plasmid replicon types. CONCLUSION The genome sequence reported herein will provide useful information for a better understanding of the genetic structure of the S. epidermidis genome in Africa.