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Infective endocarditis (IE), characterized by inflammation of the endocardial surface of the heart and its valves, results from infections caused by Staphylococcus, Streptococcus and Acinetobacter species and less commonly fungi. Acinetobacter-induced IE is a relatively rare condition with significant morbidity and mortality worldwide. Notably, its mortality rate is greater than that of endocarditis induced by the Haemophilus species, Aggregatibacter actinomycetemcomitans, Cardiobacterium hominis, Eikenella corrodens and Kingella kingae. Although it is rare, Acinetobacter-induced IE caused by A. baumannii might bring unique therapeutic challenges such as increased antibiotic resistance. Therefore, it is vital to understand perfectly the possible pathophysiologic and antibiotic resistance mechanisms adopted by A. baumannii during IE. This review discusses the probable underlying pathomechanisms involved in A. baumannii-induced IE and highlights the potential antibiotic resistance mechanisms, suggesting therapeutic targets for A. baumannii-induced IE.

The growing rate of antibiotic resistance is an important global health threat. This threat is exacerbated by the lack of safe and potent alternatives to carbapenems in addition to the slow developmental process of newer and effective antibiotics.

IncX3 and IncL plasmids have been named as catalysts advancing dissemination of

The gold standard forMycobacterium ulceransdetection is PCR due to its high accuracy in confirmation of suspected cases. But the available PCR assays are design for standard size thermocyclers which are immobile and suited for reference laboratories far away from endemic communities. This makes it a challenge to obtain immediate results for patient management. We have validated and evaluated a dried reagent-based PCR assay adapted for a handheld, battery-operated, portable thermocycler with the potential of extending diagnostics to endemic communities with limited infrastructure. The diagnostic accuracy of the assay following a multi-centre evaluation by three Buruli ulcer reference laboratories with over 300 clinical samples showed sensitivity and specificity of 100% - 97% and 100% - 94%. This assay coupled with a field-friendly extraction method fulfill almost all the target product profile of Buruli ulcer for decentralized testing at districts, health centres and community level. A key critical action for achieving the NTD Road Map 2030 target for Buruli ulcer.

Background Early diagnosis and treatment of Buruli ulcer is critical in order to avoid the debilitating effects of the disease. In this regard, the development of new diagnostic and point of care tools is encouraged. The loop-mediated isothermal amplification for the detection of Mycobacterium ulcerans represents one of the new tools with a good potential of being developed into a point of care test. There is however the need to standardize the assays, reduce sample preparation times, improve the detection/visualization system and optimize them for high-throughput screening, adaptable to low resourced laboratories. Methods In this study, we assessed two DNA extraction protocols (modified Boom and EasyNAT methods), three previously published LAMP primer sets (BURULI, MU 2404 and BU-LAMP), and compared the sensitivity and specificity of LAMP assays on three DNA amplification platforms. Results Our results show that Buruli ulcer diagnosis using primers targeting IS2404 for the LAMP method is sensitive (73.75–91.49%), depending on the DNA extraction method used. Even though the modified Boom DNA extraction method provided the best results, its instrumentation requirement prevent it from being field applicable. The EasyNAT method on the other hand is simpler and may represent the best method for DNA extraction in less resourced settings. Conclusions For further work on the development and use of LAMP tests for Buruli diagnosis, it is recommended that the BURULI sets of primers be used, as these yielded the best results in terms of sensitivity (87.50–91.49%) and specificity (89.23–100%), depending on the DNA extraction methods used.

Background Genotyping is a powerful tool for investigating outbreaks of infectious diseases and it can provide useful information such as identifying the source and route of transmission, and circulating strains involved in the outbreak. Genotyping techniques based on variable number of tandem repeats (VNTR) are instrumental in detecting heterogeneity in Mycobacterium ulcerans (MU) and also for discriminating MU from other mycobacteria species. Here, we describe and map the distribution of MU genotypes in Buruli ulcer (BU) endemic communities of the Nyong valley in Cameroon. We also tested the hypothesis of whether the suspected animal reservoirs of BU that share the human microhabitat are shedding contaminated fecal matters and saliva into their surrounding environments. Methods Environmental samples from suspected MU-risk factors and lesion swabs from human patients were sampled in BU-endemic communities and tested for the presence of MU by qPCR targeting three independent sequences (IS2404, IS2606, KR-B). Positive samples to MU were further genotyped by VNTR with confirmation by sequencing of four loci (MIRU1, Locus 6, ST1, Locus 19). Results MU was detected in environmental samples including water bodies (23%), biofilms (14%), detritus (10%), and in human patients (73%). MU genotypes D, W, and C were found both in environmental and human samples. The micro geo-distribution of MU genotypes from communities showed that genotype D is found both in environmental and human samples, while genotypes W and C are specific to environmental samples and human lesions, respectively. No obvious focal grouping of MU genotypes was observed at the community scale. An additional survey in the human microhabitat suggests that domestic and wild animals do not shed MU in their saliva and feces in sampled communities. Conclusions VNTR typing uncovered different MU genotypes circulating in the endemic communities of the Akonolinga district. A MU environmental genotype was found in patients, yet the mechanism of contamination remains to be investigated; and recovering MU in culture from the environment remains key priority to enable a better understanding of the mode of transmission of BU. We also conclude that excretions from suspected animals are unlikely to be major sources of MU in the Nyong Valley in Cameroon.

Diarrheagenic Escherichia coli (DEC), an important agent of infectious diarrhea, is constantly evolving, making its periodic monitoring necessary. However, the DEC genotypes in Ghana remain uncharacterized. We focused on characterizing the molecular serotypes, virulence factors, multilocus sequence types, and the phylogenetic relatedness among different DEC pathotypes recovered from stool samples of pediatric patients with symptoms of diarrhea from the Western region of Ghana. We detected all five common DEC pathotypes, with the majority of the isolates being enterotoxigenic E. coli (ETEC) harboring the heat-labile enterotoxin gene. The DEC strains exhibited diverse serotypic identity with novel and previously reported outbreak strains. Sequence types (ST) ST38, ST316, and ST1722 were most prevalent, and clonal complex 10 (CC10) was the most common CC. A close evolutionary distance was observed among most of the isolates. Coli surface antigen 6 was the most prevalent (44%, n = 11) ETEC-specific colonization factor. Nearly all the isolates harbored lpfA, and the frequencies of other virulence genes such as pap and cnf1 were 7.9% and 18.4%, respectively. This study provides insights into the important and novel genotypes circulating in the Western region of Ghana that should be monitored for public health.

The gold standard for detection of Mycobacterium ulcerans is PCR due to its high accuracy in confirmation of suspected cases. But the available PCR assays are designed for standard size thermocyclers which are immobile and suited for reference laboratories often located long distances from endemic communities. This makes it a challenge to obtain immediate results for patient management. We validated and evaluated a dried reagent-based PCR assay adapted for a handheld, battery-operated, portable thermocycler with the potential to extend diagnostics to endemic communities with limited infrastructure. The diagnostic accuracy of the assay following a multi-center evaluation by three Buruli ulcer reference laboratories with over 300 clinical samples showed sensitivity and specificity of 100–97% and 100–94%, respectively using centralized IS2404 quantitative PCR platform as a reference standard. This assay coupled with a field-friendly extraction method fulfill almost all the target product profiles of Buruli ulcer for decentralized testing at the district, health center and community levels; a key critical action for achieving the NTD Road Map 2030 target for Buruli ulcer.

The emergence and spread of carbapenemase-producing bacteria are serious threats to public health. We characterized two OXA-181-producing Escherichia coli isolates from pediatric patients with diar...

Introduction: Urinary tract infection represents more than 40% of all nosocomial infections of which 80% are attributed to the use of an indwelling urinary catheter. Although the device is useful medically to treat urinary retention and incontinence, its uses have also been associated with infections such as catheter-associated urinary tract infections. Objectives: To evaluate catheter-associated urinary tract infection in terms of its prevalence, pathogens causing the infection and the resistance pattern to some commonly used antibiotics. Methods: Urine and biofilm specimen were collected from 105 catheterized patients. Bacteria pathogens were identified based on colony morphology, Gram staining and other biochemical reactions. Kirby-Bauer disc diffusion method was employed to determine the antibiotic resistance pattern of the isolated pathogens. Results: The study recorded a 74.29% prevalence of catheter-associated urinary tract infection among the rarely symptomatic patients. Escherichia coli were significantly isolated (p Staphylococcus aureus. Most isolated pathogens from both samples were resistant to ciprofloxacin with the biofilm pathogens being highly resistant than the urine pathogens. K. pneumoniae from both samples showed higher resistance levels to antibiotics than E. coli and S. aureus. Conclusion: Ciprofloxacin, a commonly used antibiotic by catheterized patients turn out to be less effective against the pathogens. The use of ciprofloxacin in catheter-associated urinary tract infection treatment should therefore be given a second thought.

This study determines the antibiotic resistance pattern of extended spectrum beta-lactamase (ESBL) producing pathogens responsible for catheter associated urinary tract infection (CAUTI) and the genes associated with the resistance. The study used 35 ESBL-producing pathogens isolated from urine and biofilm found in CAUTI from June 2018 to November 2018. Pathogens were confirmed phenotypically for ESBL production with cefpodoxime combination disc kits. Antibiotic resistance was tested using the Kirby-Baurer disc diffusion method on Muller Hinton Agar. ESBL genotypes were identified with PCR. Urine isolates showed higher frequency of resistance against ciprofloxacin (94.11%), cefuroxime and ceftazidime (82.35%) and with no recorded resistance against Eertepenem (0.0%). The average resistance of the biofilm isolates ranged from 0.0% (Ertepenem) to 88.89% (Cefuroxime, Cefpodoxime, Ciprofloxacin and Trimethoprim). All the targeted genes were identified with CTX-M (40%) being the most dominant among them. The ESBL-producing pathogens showed zero resistance against Ertepenem. Ciprofloxacin and other Cephems commonly used in CAUTI treatment were shown to be less effective. The high resistance is as a result of the bacterial cells present in the biofilm with Klebsiella pneumonia exhibiting more resistance than the ESBL-producing E. coli and CTX-M-1 was identified as the most prevalent gene among the identified genotypes. Ertepenem should therefore be recommended for treatment of Catheter associated urinary tract infection. Key words: Catheter, CAUTI, extended spectrum beta-lactamase (ESBL), urine, biofilm, resistance, microorganisms.

Background Buruli ulcer is a tissue necrosis infection caused by an environmental mycobacterium called Mycobacterium ulcerans (MU). The disease is most prevalent in rural areas with the highest rates in West and Central African countries. The bacterium produces a toxin called mycolactone which can lead to the destruction of the skin, resulting in incapacitating deformities with an enormous economic and social burden on patients and their caregivers. Even though there is an effective antibiotic treatment for BU, the control and management rely on early case detection and rapid diagnosis to avert morbidities. The diagnosis of Mycobacterium ulcerans relies on smear microscopy, culture histopathology, and PCR. Unfortunately, all the current laboratory diagnostics have various limitations and are not available in endemic communities. Consequently, there is a need for a rapid diagnostic tool for use at the community health centre level to enable diagnosis and confirmation of suspected cases for early treatment. The present study corroborated the diagnostic performance and utility of fluorescent-thin layer chromatography (f-TLC) for the diagnosis of Buruli ulcer. Methodology/Principal findings The f-TLC method was evaluated for the diagnosis of Buruli ulcer in larger clinical samples than previously reported in an earlier preliminary study Wadagni et al. (2015). A total of 449 patients suspected of BU were included in the final data analysis out of which 122 (27.2%) were positive by f-TLC and 128 (28.5%) by PCR. Using a composite reference method generated from the two diagnostic methods, 85 (18.9%) patients were found to be truly infected with M. ulcerans, 284 (63.3%) were uninfected, while 80 (17.8%) were misidentified as infected or noninfected by the two methods. The data obtained was used to determine the discriminatory accuracy of the f-TLC against the gold standard IS2404 PCR through the analysis of its sensitivity, specificity, positive (+LR), and negative (–LR) likelihood ratio. The positive (PPV) and negative (NPV) predictive values, area under the receiver operating characteristic curve Azevedo et al. (2014), and diagnostic odds ratio were used to assess the predictive accuracy of the f-TLC method. The sensitivity of f-TLC was 66.4% (85/128), specificity was 88.5% (284/321), while the diagnostic accuracy was 82.2% (369/449). The AUC stood at 0.774 while the PPV, NPV, +LR, and–LR were 69.7% (85/122), 86.9% (284/327), 5.76, and 0.38, respectively. The use of the rule-of-thumb interpretation of diagnostic tests suggests that the method is good for use as a diagnostic tool. Conclusions/Significance Larger clinical samples than previously reported had been used to evaluate the f-TLC method for the diagnosis of Buruli ulcer. A sensitivity of 66.4%, a specificity of 88.5%, and diagnostic accuracy of 82.2% were obtained. The method is good for diagnosis and will help in making early clinical decisions about the patients as well as patient management and facilitating treatment decisions. However, it requires a slight modification to address the challenge of background interference and lack of automatic readout to become an excellent diagnostic tool.

Extra-intestinal pathogenic Escherichia coli (ExPEC) is one of the world’s leading causes of bloodstream infections with high mortality. Sequence type 410 (ST410) is an emerging ExPEC clone resistant to a wide range of antibiotics. In this study, we investigated the epidemiology of 21 ST410 E. coli isolates from two Ghanaian hospitals. We also investigated the isolates within a global context to provide further insight into the dissemination of this highly pathogenic clone. A phylogenetic tree of the 21 isolate genomes, along with 102 others from global collection, was constructed representing the ensuing clades and sub-clades of the ST: A/H53, B2/H24R, B3/H24Rx, and B4/H24RxC. The carbapenem-resistant sub-clade B4/H24RxC is reported to have emerged in the early 2000s when ST410 acquired an IncX3 plasmid carrying a blaOXA–181 carbapenemase gene, and a second carbapenemase gene, blaNDM–5, on a conserved IncFII plasmid in 2014. We identified, in this study, one blaOXA–181–carrying isolate belonging to B4/H24RxC sub-lineage and one carrying blaNDM–1 belonging to sub-lineage B3/H24Rx. The blaOXA–181 gene was found on a 51kb IncX3 plasmid; pEc1079_3. The majority (12/21) of our Ghanaian isolates were clustered with international strains described by previous authors as closely related strains to B4/H24RxC. Six others were clustered among the ESBL-associated sub-lineage B3/H24Rx and three with the globally disseminated sub-lineage B4/H24RxC. The results show that this highly pathogenic clone is disseminated in Ghana and, given its ability to transmit between hosts, it poses a serious threat and should be monitored closely.

Extra-intestinal pathogenic Escherichia coli (ExPEC) is one of the world’s leading causes of bloodstream infections with high mortality. Sequence type 410 (ST410) is an emerging ExPEC clone resistant to a wide range of antibiotics. In this study, we investigated the epidemiology of 21 ST410 E. coli isolates from two Ghanaian hospitals. We also investigated the isolates within a global context to provide further insight into the dissemination of this highly pathogenic clone. A phylogenetic tree of the 21 isolate genomes, along with 102 others from global collection, was constructed representing the ensuing clades and sub-clades of the ST: A/H53, B2/H24R, B3/H24Rx, and B4/H24RxC. The carbapenem-resistant sub-clade B4/H24RxC is reported to have emerged in the early 2000s when ST410 acquired an IncX3 plasmid carrying a blaOXA–181 carbapenemase gene, and a second carbapenemase gene, blaNDM–5, on a conserved IncFII plasmid in 2014. We identified, in this study, one blaOXA–181–carrying isolate belonging to B4/H24RxC sub-lineage and one carrying blaNDM–1 belonging to sub-lineage B3/H24Rx. The blaOXA–181 gene was found on a 51kb IncX3 plasmid; pEc1079_3. The majority (12/21) of our Ghanaian isolates were clustered with international strains described by previous authors as closely related strains to B4/H24RxC. Six others were clustered among the ESBL-associated sub-lineage B3/H24Rx and three with the globally disseminated sub-lineage B4/H24RxC. The results show that this highly pathogenic clone is disseminated in Ghana and, given its ability to transmit between hosts, it poses a serious threat and should be monitored closely.

The emergence and spread of carbapenemase-producing bacteria are serious threats to public health. We characterized two OXA-181-producing Escherichia coli isolates from pediatric patients with diarrhea from Ghana. blaOXA-181 was localized on the self-conjugative IncX3-containing plasmid in the E. coli ST410 isolate, belonging to an emerging lineage, and an IncFIC(FII)-containing plasmid in E. coli ST940. The blaOXA-181-qnrS1 region was found on the IS26 composite transposon, which contained a 366-bp deletion in the region encoding the Rep A protein for the IncX3-containing plasmid. The IncFIC(FII) plasmid was novel and integrated with an approximately 39-kb IncX1 plasmid through conjugal transfer. Both plasmids clustered close to plasmids from Switzerland. To the best of our knowledge, this is the first report describing the presence of an IncX3 plasmid containing blaOXA-181 in strains closely related to the B4/H24RxC clade in Africa, suggesting its emergence and the need to strengthen antimicrobial resistance surveillance.

Recently, the emergence and rapid dissemination of extended-spectrum beta-lactamase (ESBL)-producing bacteria, particularly of the family Enterobacteriaceae, has posed serious healthcare challenges. Here, we determined the antimicrobial susceptibility and genetic characteristics of 164 Escherichia coli strains isolated from infected patients in two hospitals in Ghana. In total, 102 cefotaxime-resistant isolates (62.2%) were identified as ESBL-producers. Multilocus sequence typing of the ESBL-producers identified 20 different sequence types (STs) with ST131 (n = 25, 24.5%) as the dominant group. Other detected STs included ST410 (n = 21, 20.6%) and ST617 (n = 19, 18.6%). All identified ESBL-producers harbored blaCTX-M-14, blaCTX-M-15, or blaCTX-M-27, with blaCTX-M-15 (n = 96, 94.1%) being the most predominant ESBL allele. Further analysis showed that the immediate genetic environment around blaCTX-M-15 is conserved within blaCTX-M-15 containing strains. Five of the 25 ST131 isolates were clustered with clade A, one with sub-clade C1, and 19 with the dominant sub-clade C2. The results show that fluoroquinolone-resistant, blaCTX-M-14- and blaCTX- M-15-producing ESBL E. coli ST131 strains belonging to clade A and sub-clades C1 and C2 are disseminating in Ghanaian hospitals. To the best of our knowledge, this is the first report of the ST131 phylogeny in Ghana.

Data on molecular characterization of coagulase-negative staphylococci causing neonatal sepsis in low-income countries are highly limited. This report highlights the isolation of three Staphylococcus epidermidis strains, NGASs, from blood samples from neonates, with unknown transmission sources. Pathogenic factors and sources of transmission of these strains warrant further investigation., Data on molecular characterization of coagulase-negative staphylococci causing neonatal sepsis in low-income countries are highly limited. This report highlights the isolation of three Staphylococcus epidermidis non-genome assembly strains (NGASs) from blood samples from neonates with unknown transmission sources. Pathogenic factors and sources of transmission of these strains warrant further investigation.

Additional file 1. Distribution of sampled water bodies within the study sites in Akonolinga health district.

Additional file 2. Distribution of MU-positivity per sampled locality and sample type.

Multidrug resistance, especially carbapenem resistance in Acinetobacter bacteria is a global healthcare concern. However, available data on the phenotypic and genotypic characteristics of Acinetobacter isolates from West Africa, including Ghana is scanty. Our aim was to investigate the antibiotic resistance profile and genotypic characteristics of Acinetobacter isolates from Ghana and to characterize carbapenemase producers using whole-genome sequencing (WGS). A total of 36 Acinetobacter isolates collected at three hospitals in Ghana between 2016 and 2017 were analyzed. MICs were determined by commercial antibiotic plates. Acinetobacter baumannii MLST was determined using the Pasteur scheme. WGS of OXA-carbapenemase producers was performed using short- and long-read sequencing strategies. The resistance rate was highest for trimethoprim/sulfamethoxazole (n = 22; 61%). Six (16.7%) and eight (22.2%) isolates were resistant to ceftazidime and colistin, respectively. Two (5.6%) isolates were resistant and one (2.8%) isolate had intermediate sensitivity to three carbapenems. Fifteen STs were identified in 24 A. baumannii isolates including six new STs (ST1467 ∼ ST1472). ST78 was the predominant (n = 6) followed by ST1469 (n = 3). Four carbapenemase-producing A. baumannii isolates also were identified. Isogenic ST103 isolates Ab-B004d-c and Ab-D10a-a harbored bla OXA- 23 within Tn2007 on identical plasmids, pAb-B004d-c_3, and pAb-D10a-a_3. ST1472 isolate Ab-C102 and ST107 isolate Ab-C63 carried bla OXA- 58 and bla OXA- 420, a rare bla OXA- 58 variant, respectively, within novel genetic contexts. Our results show that A. baumannii isolates of diverse and unique genotypes, including OXA-carbapenemase producers, are circulating in Ghana highlighting the need for a wider surveillance of antimicrobial resistance.

Diarrheagenic Escherichia coli (DEC), an important agent of infectious diarrhea, is constantly evolving, making its periodic monitoring necessary. However, the DEC genotypes in Ghana remain uncharacterized. We focused on characterizing the molecular serotypes, virulence factors, multilocus sequence types, and the phylogenetic relatedness among different DEC pathotypes recovered from stool samples of pediatric patients with symptoms of diarrhea from the Western region of Ghana. We detected all five common DEC pathotypes, with the majority of the isolates being enterotoxigenic E. coli (ETEC) harboring the heat-labile enterotoxin gene. The DEC strains exhibited diverse serotypic identity with novel and previously reported outbreak strains. Sequence types (ST) ST38, ST316, and ST1722 were most prevalent, and clonal complex 10 (CC10) was the most common CC. A close evolutionary distance was observed among most of the isolates. Coli surface antigen 6 was the most prevalent (44%, n = 11) ETEC-specific colonization factor. Nearly all the isolates harbored lpfA, and the frequencies of other virulence genes such as pap and cnf1 were 7.9% and 18.4%, respectively. This study provides insights into the important and novel genotypes circulating in the Western region of Ghana that should be monitored for public health.

Multidrug resistance, especially carbapenem resistance in Acinetobacter bacteria is a global healthcare concern. However, available data on the phenotypic and genotypic characteristics of Acinetobacter isolates from West Africa, including Ghana is scanty. Our aim was to investigate the antibiotic resistance profile and genotypic characteristics of Acinetobacter isolates from Ghana and to characterize carbapenemase producers using whole-genome sequencing (WGS). A total of 36 Acinetobacter isolates collected at three hospitals in Ghana between 2016 and 2017 were analyzed. MICs were determined by commercial antibiotic plates. Acinetobacter baumannii MLST was determined using the Pasteur scheme. WGS of OXA-carbapenemase producers was performed using short- and long-read sequencing strategies. The resistance rate was highest for trimethoprim/sulfamethoxazole (n = 22; 61%). Six (16.7%) and eight (22.2%) isolates were resistant to ceftazidime and colistin, respectively. Two (5.6%) isolates were resistant and one (2.8%) isolate had intermediate sensitivity to three carbapenems. Fifteen STs were identified in 24 A. baumannii isolates including six new STs (ST1467 ∼ ST1472). ST78 was the predominant (n = 6) followed by ST1469 (n = 3). Four carbapenemase-producing A. baumannii isolates also were identified. Isogenic ST103 isolates Ab-B004d-c and Ab-D10a-a harbored blaOXA–23 within Tn2007 on identical plasmids, pAb-B004d-c_3, and pAb-D10a-a_3. ST1472 isolate Ab-C102 and ST107 isolate Ab-C63 carried blaOXA–58 and blaOXA–420, a rare blaOXA–58 variant, respectively, within novel genetic contexts. Our results show that A. baumannii isolates of diverse and unique genotypes, including OXA-carbapenemase producers, are circulating in Ghana highlighting the need for a wider surveillance of antimicrobial resistance.

Purpose of the Review Buruli ulcer (BU) is a necrotizing and disabling cutaneous disease caused by Mycobacterium ulcerans, one of the skin-related neglected tropical diseases (skin NTDs). This article aims to review the current knowledge of this disease and challenges ahead. Recent Findings Around 60,000 cases of BU have been reported from over 33 countries between 2002 and 2017. Encouraging findings for development of point-of-care tests for BU are being made, and its treatment is currently in the transition period from rifampicin plus streptomycin (injection) to all-oral regimen. A major recent advance in our understanding of its pathogenesis has been agreement on the mechanism of action of the major virulence toxin mycolactone in host cells, targeting the Sec61 translocon during a major step in protein biogenesis. Summary BU is distributed mainly in West Africa, but cases are also found in other parts of the world. We may be underestimating its true disease burden, due to the limited awareness of this disease. More awareness and more understanding of BU will surely contribute in enhancing our fight against this skin NTD.

Buruli ulcer (BU) is a necrotizing infection of the skin and subcutaneous tissue caused by Mycobacterium ulcerans. BU wounds may also be colonized with other microorganisms including Staphylococcus aureus. This study aimed to characterize the virulence factors of S. aureus isolated from BU patients. Previously sequenced genomes of 21 S. aureus isolates from BU patients were screened for the presence of virulence genes. The results show that all S. aureus isolates harbored on their core genomes genes for known virulence factors like alpha-hemolysin, and the a and beta-phenol soluble modulins. Besides the core genome virulence genes, mobile genetic elements (MGEs), i.e. prophages, genomic islands, pathogenicity islands and a Staphylococcal cassette chromosome (ECG) were found to carry different combinations of virulence factors, among them genes that are known to encode factors that promote immune evasion, superantigens and Panton-Valentine Leucocidin. The present observations imply That the S. aureus isolates from BU patients harbor a diverse repertoire of virulence genes that may enhance bacterial survival and persistence in the wound environment and potentially contribute to delayed wound healing.

Global dissemination of New Delhi metallo-β-lactamase (NDM)-producing bacteria has become a major health threat. However, there are few reports regarding the identification and characterisation of NDM-producing bacteria from West Africa, including Ghana. An Escherichia coli strain with resistance to meropenem was isolated from the Tamale Teaching Hospital in Ghana. Its identification and determination of antibiotic susceptibility profile were carried out using commercial systems. The antibiotic resistance mechanism was analysed by phenotypic detection kits, PCR, and DNA sequencing. Conjugation experiments, S1 nuclease pulsed field gel electrophoresis, and Southern blotting were performed. Finally, the NDM-1-harbouring plasmid was characterised using next-generation sequencing and phylogenetic analysis. The meropenem-resistant Escherichia coli strain EC2189 harboured blaNDM-1 and belonged to sequence type 410. blaNDM-1 was located on the IncHI type transferrable plasmid p2189-NDM (248,807 bp long), which co-carried multiple resistance genes, such as blaCTX-M-15, aadA1, aac(6')-Ib, sul3, dfrA12, and cmlA1. p2189-NDM phylogenetically differed from previously identified blaNDM-1-positive IncHI type plasmids. A truncated Tn125 containing blaNDM-1 was bracketed by an ISSm-1-like insertion sequence upstream and by a site-specific integrase downstream. To the best of our knowledge, we have, for the first time identified and molecularly characterised an NDM-1-producing Enterobacteriaceae strain in Ghana with blaNDM-1 that had a novel genetic structure. Our findings indicate a possibility of NDM-1 dissemination in Ghana and underscore the need for constant monitoring of carbapenemase-producing bacteria.

Background The environmental pathogen, Mycobacterium ulcerans (MU) can infect both humans and animals and cause Buruli ulcer (BU) disease. However, its mode(s) of transmission from the colonized environment to human/animal hosts remain unclear. In Australia, MU can infect both wildlife and domestic mammals. Till date, BU-like lesions have only been reported in wildlife in Africa. This warrants a thorough assessment of possible MU in domestic animals in Africa. Here, we screened roaming domesticated animals that share the human microhabitat in two different BU endemic sites, Sedje-Denou in Benin and Akonolinga in Cameroon, for MU lesions. Methodology/Principal findings We screened roaming mammals and birds across 3 endemic villages of Sedje-Denou in Southern Benin and 6 endemic villages of Akonolinga in Cameroon. After approval from relevant authorities, specimens (wound swabs and tissue fragments) were collected from animals with open or active lesion and systematically screened to detect the presence of MU though the diagnostic DNA targets IS2404, IS2606 and KR-B. Out of 397 animals surveyed in Akonolinga, 44 (11.08%) carried skin lesions and all were negative for MU DNA. For Sedje-Denou, only 25 (6.93%) out of 361 animals surveyed carried external skin lesions of which 2 (8%) were positive for MU DNA targets. These MU infected lesions were found in two different villages on a goat (abdominal part) and on a dog (nape area of the neck). Source-tracking of MU isolates within infected animal lesions was performed using VNTR genotyping and further confirmed with sequencing. One MU VNTR genotype (Z) was successfully typed from the goat lesion. The evolutionary history inferred from sequenced data revealed a clustering of animal MU isolates within isolates from human lesions. Conclusion/Significance This study describes the first report of two MU infected lesions in domestic animals in Africa. Their DNA sequence analyses show close relationship to isolates from human cases. It suggests that MU infection should be suspected in domestic hosts and these could play a role in transmission. The findings further support the hypothesis that MU is a ubiquitous environmental pathogen found in endemic areas, and probably involved in a multiple transmission pathway., Author summary Buruli ulcer (BU) remains a major Public Health problem in rural communities in sub-Saharan Africa. There are several reports of the occurrence of BU in Wildlife as well as domestic animals in Australia leading to the suggestion that animals may play a role in the transmission of MU to humans. Report of BU in animals is however scanty in Africa and no significant link has been made between BU in humans and animals. BU-like lesions were investigated in 397 and 361 roaming domestic animals respectively from Sedje-Denou and Akonolinga. Wound swabs, and tissue fragments were collected from animals with active lesions. Overall, 2 (8%) type I (

Objectives: Staphylococcus aureus infections in burn patients can lead to serious complications and death. The frequency of S. aureus infection is high in low-and middle-income countries presumably due to limited resources, misuse of antibiotics and poor infection control. The objective of the present study was to apply population genomics to precisely define, for the first time, the transmission of antibiotic resistant S. aureus in a resource-limited setting in sub-Saharan Africa.Methods: Staphylococcus aureus surveillance was performed amongst burn patients and healthcare workers during a 7-months survey within the burn unit of the Korle Bu Teaching Hospital in Ghana.Results: Sixty-six S. aureus isolates (59 colonizing and 7 clinical) were obtained from 31 patients and 10 healthcare workers. Twenty-one of these isolates were ST250-IV methicillin-resistant S. aureus (MRSA). Notably, 25 (81%) of the 31 patients carried or were infected with S. aureus within 24 h of admission. Genome comparisons revealed six distinct S. aureus clones circulating in the burn unit, and demonstrated multiple transmission events between patients and healthcare workers. Further, the collected S. aureus isolates exhibited a wide range of genotypic resistances to antibiotics, including trimethoprim (21%), aminoglycosides (33%), oxacillin (33%), chloramphenicol (50%), tetracycline (59%) and fluoroquinolones (100%).Conclusion: Population genomics uncovered multiple transmission events of S. aureus, especially MRSA, within the investigated burn unit. Our findings highlight lapses in infection control and prevention, and underscore the great importance of active surveillance to protect burn victims against multi-drug resistant pathogens in resource-limited settings.

Objectives: Staphylococcus aureus infections in burn patients can lead to serious complications and death. The frequency of S. aureus infection is high in low- and middle-income countries presumably due to limited resources, misuse of antibiotics and poor infection control. The objective of the present study was to apply population genomics to precisely define, for the first time, the transmission of antibiotic resistant S. aureus in a resource-limited setting in sub-Saharan Africa. Methods: Staphylococcus aureus surveillance was performed amongst burn patients and healthcare workers during a 7-months survey within the burn unit of the Korle Bu Teaching Hospital in Ghana. Results: Sixty-six S. aureus isolates (59 colonizing and 7 clinical) were obtained from 31 patients and 10 healthcare workers. Twenty-one of these isolates were ST250-IV methicillin-resistant S. aureus (MRSA). Notably, 25 (81%) of the 31 patients carried or were infected with S. aureus within 24 h of admission. Genome comparisons revealed six distinct S. aureus clones circulating in the burn unit, and demonstrated multiple transmission events between patients and healthcare workers. Further, the collected S. aureus isolates exhibited a wide range of genotypic resistances to antibiotics, including trimethoprim (21%), aminoglycosides (33%), oxacillin (33%), chloramphenicol (50%), tetracycline (59%) and fluoroquinolones (100%). Conclusion: Population genomics uncovered multiple transmission events of S. aureus, especially MRSA, within the investigated burn unit. Our findings highlight lapses in infection control and prevention, and underscore the great importance of active surveillance to protect burn victims against multi-drug resistant pathogens in resource-limited settings.

Background: In developing countries, hospitalized burn victims are at high risk of nosocomial infections caused by Staphylococcus aureus. Risk factors include poor infection control practices, prolonged hospitalisation and limited capacity for laboratory microbiological analyses. These problems are compounded by widespread use of antibiotics that drives the spread of multidrug resistant bacteria.Methods: During the study period (November 2014-June 2015), nasal and invasive S. aureus isolates were collected consecutively from patients and healthcare workers (HCWs) within the burn unit of the Reconstructive Plastic Surgery and Burn Center of Korle Bu Teaching Hospital in Ghana. Antibiotic prescription, antibiotic susceptibility and bacterial typing were used to assess antibiotic pressure, antibiotic resistance, and possible transmission events among patients and HCWs.Results: Eighty S. aureus isolates were obtained from 37 of the 62 included burn patients and 13 of the 29 HCWs. At admission, 50% of patients carried or were infected with S. aureus including methicillin resistant S. aureus (MRSA). Antibiotic use per 100 days of hospitalization was high (91.2 days), indicating high selective pressure for resistant pathogens. MRSA isolates obtained from 11 patients and one HCW belonged to the same spa-type t928 and multi-locus sequence type 250, implying possible transmission events. A mortality rate of 24% was recorded over the time of admission in the burn unit.Conclusion: This study revealed a high potential for MRSA outbreaks and emergence of resistant pathogens amongst burn patients due to lack of patient screening and extended empirical use of antibiotics. Our observations underscore the need to implement a system of antibiotic stewardship and infection prevention where microbiological diagnostics results are made available to physicians for timely and appropriate patient treatment.

Buruli ulcer is an indolent, slowly progressing necrotizing disease of the skin caused by infection with Mycobacterium ulcerans . In the present study, we applied a redesigned technique to a vast panel of M. ulcerans disease isolates and clinical samples originating from multiple African disease foci in order to (i) gain fundamental insights into the population structure and evolutionary history of the pathogen and (ii) disentangle the phylogeographic relationships within the genetically conserved cluster of African M. ulcerans . Our analyses identified 23 different African insertion sequence element single nucleotide polymorphism (ISE-SNP) types that dominate in different areas where Buruli ulcer is endemic. These ISE-SNP types appear to be the initial stages of clonal diversification from a common, possibly ancestral ISE-SNP type. ISE-SNP types were found unevenly distributed over the greater West African hydrological drainage basins. Our findings suggest that geographical barriers bordering the basins to some extent prevented bacterial gene flow between basins and that this resulted in independent focal transmission clusters associated with the hydrological drainage areas. Different phylogenetic methods yielded two well-supported sister clades within the African ISE-SNP types. The ISE-SNP types from the “pan-African clade” were found to be widespread throughout Africa, while the ISE-SNP types of the “Gabonese/Cameroonian clade” were much rarer and found in a more restricted area, which suggested that the latter clade evolved more recently. Additionally, the Gabonese/Cameroonian clade was found to form a strongly supported monophyletic group with Papua New Guinean ISE-SNP type 8, which is unrelated to other Southeast Asian ISE-SNP types.

Identifying the source reservoirs ofMycobacterium ulceransis key to understanding the mode of transmission of this pathogen and controlling the spread of Buruli ulcer (BU). In Australia, the native possum can harborM. ulceransin its gastrointestinal tract and shed high concentrations of the bacteria in its feces. To date, an analogous animal reservoir in Africa has not been identified. Here we tested the hypothesis that common domestic animals in BU endemic villages of Ghana are reservoir species analogous to the Australian possum. Using linear-transects at 10-meter intervals, we performed systematic fecal surveys across four BU endemic villages and one non-endemic village in the Asante Akim North District of Ghana. One hundred and eighty fecal specimens from a single survey event were collected and analyzed by qPCR for theM. ulceransdiagnostic DNA targets IS2404and KR-B. Positive and negative controls performed as expected but all 180 test samples were negative. This structured snapshot survey suggests that common domestic animals living in and around humans do not shedM. ulceransin their feces. We conclude that, unlike the Australian native possum, domestic animals in rural Ghana are unlikely to be major reservoirs ofM. ulcerans.

To optimize Buruli ulcer (BU) microscopic diagnosis, we compared two smear preparation methods from tissue specimens: smears made with tissue suspension after grinding and smears made directly with unground tissue. We also compared two smear staining methods: auramine and Ziehl-Neelsen (ZN). IS 2404-PCR was used as reference method. One hundred and thirty-one surgical tissue specimens from patients suspected of having BU were analyzed. Both smear preparation methods and both staining methods were equivalent in any combination. Thus we recommend ZN stained smears of unground tissue for peripheral treatment centres.

We report a case of a four-year-old Angolan boy with the edematous form of Buruli ulcer on the face and scalp, who was treated at a rural hospital in the Bas-Congo Province, Democratic Republic of Congo. Treatment consisted of a series of surgical interventions and antimycobacterial chemotherapy (rifampin and ciprofloxacin) for two months. This case demonstrates the diagnostic and management difficulties of an edematous lesion of BU on the face and suggests an enhancement of healing and limitation of extent of excision by specific antibiotherapy. The outcome in this patient also underscores the importance of prompt referral of suspected cases and training of health professionals in the early diagnosis of BU.

In recent years, numerous IS2404 positive mycobacteria have been identified, compromising the detection of Mycobacterium ulcerans. In this study, variable number of tandem repeats (VNTR) analysis was successfully applied on cultures and tissue specimens to differentiate all currently known IS2404 positive mycobacteria from M. ulcerans.

Mycobacterium liflandii causes a fatal frog disease in captive anurans. Here we report, to our knowledge, the first epizootic of mycobacteriosis in a European colony of clawed frogs (Silurana tropicalis), previously imported from a United States biologic supply company. Our findings suggest the emerging potential of this infection through international trade.

Background Buruli ulcer (BU) is a skin infection caused by Mycobacterium ulcerans. The wounds of most BU patients are colonized with different microorganisms, including Staphylococcus aureus. Methodology This study investigated possible patient-to-patient transmission events of S. aureus during wound care in a health care center. S. aureus isolates from different BU patients with overlapping visits to the clinic were whole-genome sequenced and analyzed by a gene-by-gene approach using SeqSphere+ software. In addition, sequence data were screened for the presence of genes that conferred antibiotic resistance. Principal Findings SeqSphere+ analysis of whole-genome sequence data confirmed transmission of methicillin resistant S. aureus (MRSA) and methicillin susceptible S. aureus among patients that took place during wound care. Interestingly, our sequence data show that the investigated MRSA isolates carry a novel allele of the fexB gene conferring chloramphenicol resistance, which had thus far not been observed in S. aureus., Author Summary Buruli ulcer (BU) is a skin infection caused by Mycobacterium ulcerans. The wounds of most BU patients are colonized with different microorganisms, including Staphylococcus aureus. This study investigated patient-to-patient transmission events during wound care in a health care center. S. aureus isolates from patients who visited the health center at the same time points were analyzed using whole-genome sequencing. Analysis of sequence data confirmed transmission of methicillin resistant S. aureus and methicillin susceptible S. aureus among patients that took place during wound care.

Introduction Mycobacterium ulcerans infection, known as Buruli ulcer, is a disease of the skin and subcutaneous tissues which is an important but neglected tropical disease with its major impact in rural parts of West and Central Africa where facilities for diagnosis and management are poorly developed. We evaluated fluorescent thin layer chromatography (f-TLC) for detection of mycolactone in the laboratory using samples from patients with Buruli ulcer and patients with similar lesions that gave a negative result on PCR for the IS2404 repeat sequence of M. ulcerans Methodology/Principal findings Mycolactone and DNA extracts from fine needle aspiration (FNA), swabs and biopsy specimen were used to determine the sensitivity and specificity of f-TLC when compared with PCR for the IS2404. For 71 IS2404 PCR positive and 28 PCR negative samples the sensitivity was 73.2% and specificity of 85.7% for f-TLC. The sensitivity was similar for swabs (73%), FNAs (75%) and biopsies (70%). Conclusions We have shown that mycolactone can be detected from M. ulcerans infected skin tissue by f-TLC technique. The technique is simple, easy to perform and read with minimal costs. In this study it was undertaken by a member of the group from each endemic country. It is a potentially implementable tool at the district level after evaluation in larger field studies., Author Summary Mycobacterium ulcerans infection, known as Buruli ulcer, is a disease that affects the skin and underlying tissues. The organism responsible for the infection produces a potent toxin called mycolactone that causes extensive skin damage. Easy to perform and cheaper techniques are needed for diagnostic confirmation. We have evaluated fluorescent thin layer chromatography (fTLC) for detection of mycolactone in skin samples from patients with Buruli ulcer comparing them with samples from similar non-Buruli ulcer lesions that gave a negative result in the standard polymerase chain reaction (PCR) test for M. ulcerans. Fluorescent TLC had sensitivity of 73.2% and specificity of 85.7% when compared with PCR whether the skin sample was a swab, a biopsy or a fine needle aspirate. This study shows that mycolactone can be detected reliably from M. ulcerans infected skin tissue by the simple, low cost technique of fluorescent thin layer chromatography that could be developed for point of care use. It requires further evaluation in countries where Buruli ulcer disease is endemic.

Efforts to control the spread of Buruli ulcer--an emerging ulcerative skin infection caused by Mycobacterium ulcerans--have been hampered by our poor understanding of reservoirs and transmission. To help address this issue, we compared whole genomes from 18 clinical M. ulcerans isolates from a 30 km2 region within the Asante Akim North District, Ashanti region, Ghana, with 15 other M. ulcerans isolates from elsewhere in Ghana and the surrounding countries of Ivory Coast, Togo, Benin and Nigeria. Contrary to our expectations of finding minor DNA sequence variations among isolates representing a single M. ulcerans circulating genotype, we found instead two distinct genotypes. One genotype was closely related to isolates from neighbouring regions of Amansie West and Densu, consistent with the predicted local endemic clone, but the second genotype (separated by 138 single nucleotide polymorphisms [SNPs] from other Ghanaian strains) most closely matched M. ulcerans from Nigeria, suggesting another introduction of M. ulcerans to Ghana, perhaps from that country. Both the exotic genotype and the local Ghanaian genotype displayed highly restricted intra-strain genetic variation, with less than 50 SNP differences across a 5.2 Mbp core genome within each genotype. Interestingly, there was no discernible spatial clustering of genotypes at the local village scale. Interviews revealed no obvious epidemiological links among BU patients who had been infected with identical M. ulcerans genotypes but lived in geographically separate villages. We conclude that M. ulcerans is spread widely across the region, with multiple genotypes present in any one area. These data give us new perspectives on the behaviour of possible reservoirs and subsequent transmission mechanisms of M. ulcerans. These observations also show for the first time that M. ulcerans can be mobilized, introduced to a new area and then spread within a population. Potential reservoirs of M. ulcerans thus might include humans, or perhaps M. ulcerans-infected animals such as livestock that move regularly between countries.

Efforts to control the spread of Buruli ulcer – an emerging ulcerative skin infection caused by Mycobacterium ulcerans - have been hampered by our poor understanding of reservoirs and transmission. To help address this issue, we compared whole genomes from 18 clinical M. ulcerans isolates from a 30km2 region within the Asante Akim North District, Ashanti region, Ghana, with 15 other M. ulcerans isolates from elsewhere in Ghana and the surrounding countries of Ivory Coast, Togo, Benin and Nigeria. Contrary to our expectations of finding minor DNA sequence variations among isolates representing a single M. ulcerans circulating genotype, we found instead two distinct genotypes. One genotype was closely related to isolates from neighbouring regions of Amansie West and Densu, consistent with the predicted local endemic clone, but the second genotype (separated by 138 single nucleotide polymorphisms [SNPs] from other Ghanaian strains) most closely matched M. ulcerans from Nigeria, suggesting another introduction of M. ulcerans to Ghana, perhaps from that country. Both the exotic genotype and the local Ghanaian genotype displayed highly restricted intra-strain genetic variation, with less than 50 SNP differences across a 5.2Mbp core genome within each genotype. Interestingly, there was no discernible spatial clustering of genotypes at the local village scale. Interviews revealed no obvious epidemiological links among BU patients who had been infected with identical M. ulcerans genotypes but lived in geographically separate villages. We conclude that M. ulcerans is spread widely across the region, with multiple genotypes present in any one area. These data give us new perspectives on the behaviour of possible reservoirs and subsequent transmission mechanisms of M. ulcerans. These observations also show for the first time that M. ulcerans can be mobilized, introduced to a new area and then spread within a population. Potential reservoirs of M. ulcerans thus might include humans, or perhaps M. ulcerans-infected animals such as livestock that move regularly between countries., Author Summary In this study we use the power of whole genome sequence comparisons to track the spread of Mycobacterium ulcerans, the causative agent of Buruli ulcer, through several villages in the Ashanti region of Ghana, providing new insights on the behaviour of this enigmatic and emerging pathogen.