Comparative evaluation of analytical pipelines for illumina short- and nanopore long-read 16S rRNA gene amplicon sequencing with mock microbial communities.

Utility of a recently developed long-read pipeline, Emu, was assessed using an expectation-maximization algorithm for accurate read classification. We compared it to conventional short- and long-read pipelines, using well-characterized mock bacterial samples. Our findings highlight the necessity of appropriate data-processing for taxonomic descriptions, expanding our understanding of the precise microbiome.

Types A and F Clostridium perfringens in healthcare wastewater from Ghana.

IMPORTANCE: Clostridium perfringens causes gas gangrene and food poisoning in humans, and monitoring this bacterium is important for public health. Although whole-genome sequencing is useful to comprehensively understand the virulence, resistome, and global genetic relatedness of bacteria, limited genomic data from environmental sources and developing countries hamper our understanding of the richness of the intrinsic genomic diversity of this pathogen. Here, we successfully accumulated the genetic data on C. perfringens strains isolated from hospital effluent and provided the first evidence that predicted pathogenic C. perfringens may be disseminated in the clinical environment in Ghana. Our findings suggest the importance of risk assessment in the environment as well as the clinical setting to mitigate the potential outbreak of C. perfringens food poisoning in Ghana.

Novel insights into genetic characteristics of blaGES-encoding plasmids from hospital sewage.

Introduction: The prevalence of Guiana extended-spectrum (GES)-type carbapenemase producers is increasing worldwide, and hospital water environments are considered as potential reservoirs. However, the genetic features underlying this resistance are not yet fully understood. This study aimed to characterize blaGES-encoding plasmids from a single-hospital sewage sample in Japan. Methods: Carbapenemase producers were screened using carbapenemase-selective agar and polymerase chain reaction. Whole-genome sequencing analyzes were performed on the carbapenemase-producing isolates. Results: Eleven gram-negative bacteria (four Enterobacter spp., three Klebsiella spp., three Aeromonas spp., and one Serratia spp.) with blaGES-24 (n = 6), blaGES-6 (n = 4), and blaGES-5 (n = 1) were isolated from the sewage sample. Five blaGES-24 and a blaGES-5 were localized in IncP-6 plasmids, whereas three blaGES-6 plasmids were localized in IncC plasmids with IncF-like regions. The remaining blaGES-6 and blaGES-24 were, respectively, localized on IncFIB-containing plasmids with IncF-like regions and a plasmid with an IncW-like replication protein. The IncP-6 and IncW-like plasmids had a close genetic relationship with plasmids from Japan, whereas the IncC/IncF-like and IncFIB/IncF-like plasmids were closely related to those from the United States and Europe. All blaGES genes were located on the class 1 integron cassette of the Tn3 transposon-related region, and the IncC/IncF-like plasmid carried two copies of the integron cassette. Eight of the eleven blaGES-encoding plasmids contained toxin-antitoxin system genes. Discussion: The findings on the plasmids and the novel genetic content from a single wastewater sample extend our understanding regarding the diversity of resistance and the associated spread of blaGES, suggesting their high adaptability to hospital effluents. These findings highlight the need for the continuous monitoring of environmental GES-type carbapenemase producers to control their dissemination.

Klebsiella Species and Enterobacter cloacae Isolates Harboring blaOXA-181 and blaOXA-48: Resistome, Fitness Cost, and Plasmid Stability.

IncX3 and IncL plasmids have been named as catalysts advancing dissemination of blaOXA-181 and blaOXA-48 genes. However, their impact on the performance of host cells is vastly understudied. Genetic characteristics of blaOXA-48- and blaOXA-181-containing Klebsiella pneumoniae (EFN299), Klebsiella quasipneumoniae (EFN262), and Enterobacter cloacae (EFN743) isolated from clinical samples in a Ghanaian hospital were investigated by whole-genome sequencing. Transfer of plasmids by conjugation and electroporation, plasmid stability, fitness cost, and genetic context of blaOXA-48, blaOXA-181, and blaDHA-1 were assessed. blaOXA-181 was carried on two IncX3 plasmids, an intact 51.5-kb IncX3 plasmid (p262-OXA-181) and a 45.3-kb IncX3 plasmid (p743-OXA-181) without replication protein sequence. The fluoroquinolone-resistant gene qnrS1 region was also excised, and unlike in p262-OXA-181, the blaOXA-181 drug-resistant region was not found on a composite transposon. blaOXA-48 was carried on a 74.6-kb conjugative IncL plasmid with unknown ~10.9-kb sequence insertion. This IncL plasmid proved to be highly transferable, with a conjugation efficiency of 1.8 × 10-2. blaDHA-1 was present on an untypeable 22.2 kb genetic structure. Plasmid stability test revealed plasmid loss rate between 4.3% and 12.4%. The results also demonstrated that carriage of IncX3-blaOXA-181 or IncL-blaOXA-48 plasmids was not associated with any fitness defect, but rather an enhanced competitive ability of host cells. This study underscores the significant contribution of IncX3 and IncL plasmids in the dissemination of resistance genes and their efficient transfer calls for regular monitoring to control the expansion of resistant strains. IMPORTANCE 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. Infections by carbapenem-resistant Gram-negative bacteria are becoming almost untreatable, leading to poor clinical outcomes and high mortality rates. OXA-48-like carbapenemases are one of the most widespread carbapenemases accounting for resistance among Enterobacteriaecae. We characterized OXA-48- and OXA-181-producing Enterobacteriaecae to gain insights into the genetic basis and mechanism of resistance to carbapenems. Findings from the study showed that the genes encoding these enzymes were carried on highly transmissible plasmids, one of which had sequences absent in other similar plasmids. This implies that mobile genetic elements are important players in the dissemination of resistance genes. Further characterization of this plasmid is warranted to determine the role of this sequence in the spread of resistance genes.

Penicillin- and Ciprofloxacin-Resistant Invasive Neisseria meningitidis Isolates from Japan.

Neisseria meningitidis causes a life-threatening invasive meningococcal disease (IMD). Isolates resistant to antibiotics, such as penicillin, ceftriaxone, and ciprofloxacin that are recommended for the treatment of IMD patients and their close contacts have been serious public health concerns globally. However, susceptibility profiles to critically important antibiotics and the genetic characteristics of isolates possessing antibiotic resistance are extremely limited as IMD incidence is low in Japan. We assessed the susceptibility profiles of 87 randomly selected, sterile site-derived N. meningitidis strains isolated from hospitals nationwide, recovered between April 1998 and March 2018 in Japan, to seven antibiotics. As a result, we demonstrated, for the first time, that the isolates remained highly susceptible to ceftriaxone, meropenem, azithromycin, ciprofloxacin, chloramphenicol, and rifampin, but not to penicillin. We then characterized the genetic relatedness of six penicillin- and/or ciprofloxacin-resistant isolates obtained in this study with global 112 genomes using core-genome phylogenetic analysis. These results provide the first evidence that invasive lineages such as a penicillin-resistant serogroup W, sequence type (ST)-11 clonal complex (CC), and a ciprofloxacin-resistant serogroup B/C, ST-4821 CC that is considered as a global threat, have been sporadically identified in Japan. Our findings highlight the need to monitor antibiotic resistance in clinical isolates of N. meningitidis, thereby preventing the spread of antibiotic-resistant invasive lineages and maintaining effective treatment for IMD patients and their close contacts. IMPORTANCE Although antibiotics such as penicillin and ceftriaxone can treat invasive meningococcal disease (IMD), the emergence and spread of antibiotic-resistant Neisseria meningitidis have become a global concern. To provide effective treatment, including chemoprophylaxis to IMD patients and their close contacts, we highlighted the importance of recognizing the antibiotic resistance and genetic features of N. meningitidis isolates.

Insights and genetic features of extended-spectrum beta-lactamase producing Escherichia coli isolates from two hospitals in Ghana.

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.

Possible Dissemination of Escherichia coli Sequence Type 410 Closely Related to B4/H24RxC in Ghana.

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 bla OXA- 181 carbapenemase gene, and a second carbapenemase gene, bla NDM- 5, on a conserved IncFII plasmid in 2014. We identified, in this study, one bla OXA- 181-carrying isolate belonging to B4/H24RxC sub-lineage and one carrying bla NDM- 1 belonging to sub-lineage B3/H24Rx. The bla OXA- 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.

Emergence of oxacillinase-181 carbapenemase-producing diarrheagenic Escherichia coli in Ghana.

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.

Virulence Profiles of Diarrheagenic Escherichia coli Isolated from the Western Region of Ghana.

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.

Prevalence and Characterization of Carbapenem-Hydrolyzing Class D ß-Lactamase-Producing Acinetobacter Isolates From Ghana.

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.

Molecular characterisation of the NDM-1-encoding plasmid p2189-NDM in an Escherichia coli ST410 clinical isolate from Ghana.

Global dissemination of New Delhi metallo-ß-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.