Genomic features of an isolate of Empedobacter falsenii harbouring a novel variant of metallo-ß-lactamase, blaEBR-4 gene.

Empedobacter falsenii is an emerging opportunistic pathogen that has been occasionally implicated in various human infections. In this study, we described the genomic features of a multidrug resistant E. falsenii Q1655 obtained from a patient attending a public hospital in Sokoto, northwest Nigeria. The isolate, E. falsenii Q1655, was isolated from the stool sample of a patient in Sokoto, Nigeria. The identity of the isolate was confirmed by MALDITOF-MS. The disc diffusion test and modified Carba-NP test were used for phenotypic antibiotic susceptibility test and carbapenemase enzyme production test, respectively. The whole genome of the strain was sequenced using the Illumina MiSeq technique. Resistome analysis was done by annotation of the WGS against the ARG-ANNOT database. The isolate was resistant to all ß-lactam antibiotics with the exception of cefepime. The MICs of imipenem and ertapenem as determined by E-test were 12 µg/ml and 2 µg/ml, respectively. Modified Carba NP test showed that the strain was carbapenemase producing. Resistome analysis revealed the presence of a novel metallo-ß-lactamase, a chromosomal blaEBR-4, which exhibited 94.92% and 97.02% nucleotide and protein sequence identities respectively with blaEBR-3 gene of E. falsenii 174,820. Seven and eight amino-acid substitutions were observed with the blaEBR-1 and blaEBR-2, respectively. We reported the first isolation and genomic description of an extensively drug resistant isolate of Empedobacter falsenii in Nigeria. This report broadens our knowledge of carbapenem resistance in E. falsenii and it will serve as a useful guide in the development of antibiotic use policy.

Molecular Characterization of Clinical Carbapenem-Resistant Enterobacteriaceae Isolates from Sétif, Algeria.

This study aimed to determine the incidence and the molecular mechanisms of carbapenem-resistant Enterobacteriaceae in patients from the Sétif University Hospital, Algeria. Nonduplicate clinical bacterial isolates recovered from patients attending the University Hospital of Sétif were collected between April and October 2018. Species identification was performed by MALDI-TOF/MS (matrix-assisted laser desorption ionization-time of flight mass spectrometry) method. The susceptibility of the isolates to carbapenems was determined using the disc diffusion method. The carbapenem resistant isolates were screened for the presence of common carbapenemase genes (blaKPC, blaOXA-48, blaVIM, blaIMP, and blaNDM) and extended-spectrum ß-lactamase (blaCTX, blaTEM, and blaSHV) using PCR and sequencing technique. A total of 123 nonrepetitive Enterobacteriaceae isolates were obtained. Klebsiella pneumoniae (n = 52/42.28%), Escherichia coli (n = 24/19.51%), and Enterobacter cloacae (n = 19/15.45%) were the most prevalent species. The Carba-NP test showed that 6 out of 123 isolates carried carbapenemase enzymes. OXA-48 was found in five isolates (four K. pneumoniae and one E. coli) and NDM-5 in one E. cloacae isolate. We reported for the first time in Algeria the presence of NDM-5 carbapenemase enzyme in a clinical E. cloacae isolate.

Bhargavaea massiliensis sp. nov. and Dietzia massiliensis sp. nov., Novel Bacteria Species Isolated from Human Urine Samples in Nigeria.

Two novel bacteria species designated Marseille-Q1000T and Marseille-Q0999T were isolated from urine samples of patients in Sokoto, Northwest-Nigeria. They were Gram-positive bacteria and belong to two different genera, Bhargavaea and Dietzia. The genome size and G + C content of Marseille-Q1000T and Marseille-Q0999T were 3.07 and 3.51 Mbp with 53.8 and 71.0 mol% G + C content, respectively. The strains exhibited unique phenotypic and genomic features that are substantially different from previously known bacterial species with standing in nomenclature. On the basis of the phenotypic, phylogenetic and genomic characteristics, strains Marseille-Q0999T (= CSURQ0999 = DSM 112394) and Marseille-Q1000T (= CSURQ1000 = DSM 112384) were proposed as the type strains of Bhargavaea massiliensis sp. nov., and Dietzia massiliensis sp. nov., respectively.

First Genome Description of Providencia vermicola Isolate Bearing NDM-1 from Blood Culture.

In this paper, we describe the first complete genome sequence of Providencia vermicola species, a clinical multidrug-resistant strain harboring the New Delhi Metallo-ß-lactamase-1 (NDM-1) gene, isolated at the Kinshasa University Teaching Hospital, in Democratic Republic of the Congo. Whole genome sequencing of an imipenem-resistant clinical Gram-negative P. vermicola P8538 isolate was performed using MiSeq and Gridion, and then complete genome analysis, plasmid search, resistome analysis, and comparative genomics were performed. Genome assembly resulted in a circular chromosome sequence of 4,280,811-bp and 40.80% GC and a circular plasmid (pPV8538_NDM-1) of 151,684-bp and 51.93%GC, which was identified in an Escherichia coli P8540 strain isolated in the same hospital. Interestingly, comparative genomic analysis revealed multiple sequences acquisition within the P. vermicola P8538 chromosome, including three complete prophages, a siderophore biosynthesis NRPS cluster, a Type VI secretion system (T6SS), a urease gene cluster, and a complete Type-I-F CRISPR-Cas3 system. Β-lactamase genes, including blaCMY-6 and blaNDM-1, were found on the recombinant plasmid pPV8538_NDM-1, in addition to other antibiotic resistance genes such as rmtC, aac6'-Ib3, aacA4, catA1, sul1, aac6'-Ib-cr, tetA, and tetB. Genome comparison with Providencia species revealed 82.95% of average nucleotide identity (ANI), with P. stuartii species exhibiting 90.79% of proteome similarity. We report the first complete genome of P. vermicola species and for the first time the presence of the blaNDM-1 gene in this species. This work highlights the need to improve surveillance and clinical practices in DR Congo in order to reduce or prevent the spread of such resistance.

First whole genome sequence of Paenalcaligenes suwonensis bearing blaVIM-5 Metallo-ß-lactamase: A clinical isolate responsible for acute gastroenteritis.

Carbapenemase-producing Alcaligenes species has been described in only few studies, with none so far from the African continent. Here, we report the whole genome sequence of Peanalcaligenes suwonensis bearing blaVIM-5 metallo-ß-lactamase and first detection of carbapenemase producing Alcaligenes faecalis isolated from patients attending tertiary healthcare facilities in Nigeria. The isolates were identified by MALDI-TOF Mass Spectrometry. Antibiotic susceptibility assay, modified Carba NP test and genomic investigation revealed that two isolates of Alcaligenes faecalis and an isolate of Paenalcaligenes suwonensis harboured blaVIM-5 gene. The genome sequence analysis of the P. suwonensis 191B isolate, responsible for acute gastroenteritis, reveal the presence of 18 antibiotic resistance genes coding for resistance to five different classes of antibiotics. Three of the genes (blaOXA-368, blaCARB-4 and blaVIM-5) codes for resistance to ß-lactam antibiotics. To our best knowledge, we describe here the first genome sequence of P. suwonensis species and the first detection of class B carbapenemase blaVIM-5 in a clinical isolate of P. suwonensis species and Alcaligenes faecalis in Nigeria. The finding of this study is of concern, as lateral dissemination of the genes into clinically important Gram-negative pathogens is highly likely.

Current status of resistance to antibiotics in the Democratic Republic of the Congo: A review.

A review of literature was conducted to assess the prevalence and mechanisms of antibiotic resistance to date, mainly to ß-lactam antibiotics, cephalosporins, carbapenems, colistin, and tigecycline in the Democratic Republic of the Congo (DRC). English and French publications were listed and analysed using PubMed/Medline, Google Scholar, and African Journals database between 1 January 1990 and 31 December 2019. For the 30 published articles found: (1) bacterial resistance to antibiotics concerned both Gram-negative and Gram-positive bacteria; (2) multidrug resistance prevalence was the same in half of Streptococcus pneumoniae isolates; (3) a worrying prevalence of methicillin-resistant Staphylococcus aureus (MRSA) was noted, which is associated with co-resistance to several other antibiotics; and (4) resistance to third-generation cephalosporins was very high in Enterobacteriaceae, mainly because of blaCTX-M-1 group and blaSHV genes. Data on carbapenem and colistin resistance were not available in DRC until recently. Further work is required to set up a surveillance system for antibiotic resistance in DRC.

High prevalence of multidrug-resistant Gram-negative bacterial infections in Northwest Nigeria.

INTRODUCTION: There is limited data on the prevalence and antibiotic susceptibility profile of Gram-negative bacteria in northwest Nigeria. This study thus aimed to investigate the prevalence of multidrug resistant Gram-negative bacterial infections among patients in two healthcare facilities in Sokoto, northwest Nigeria. METHODS: A total of 735 non-duplicate clinical bacterial isolates were collected between January and July 2019, from among specimens processed by the diagnostic microbiological laboratory of the two hospitals. The isolates were identified using MALDI-TOF mass spectrometry and tested against a panel of sixteen (16) antibiotics using the current EUCAST guidelines. RESULTS: Of the 735 randomly selected bacterial isolates, 397 (54.0%) yielded Gram-negative bacteria. In the two hospitals, E. coli 104 (26.2%) and Klebsiella spp. 58 (14.6%) were the most common Gram-negative pathogens implicated in all infections. Overall, the isolates exhibited moderate to high resistance to all tested antibiotics, the lowest was observed against amikacin (7.1%). The phenotypic test for ESBL and carbapenemase enzymes showed that 48 (24.6%) and 15 (32.6%) of the isolates were positive, with 88.9% of the isolates being multidrug resistant. CONCLUSIONS: The study documents prevalent high multidrug resistant Gram-negative bacterial infections, predominantly caused by E. coli and K. pneumoniae in Sokoto, northwest Nigeria. The isolates were mostly MDR and exhibited ESBL and carbapenemase activities. The findings of this study call for urgent implementation of infection control measures and antibiotic stewardship in our hospitals so as to limit the spread of antibiotic-resistant bacteria in our healthcare facilities.

Development of real-time PCR assay allowed describing the first clinical Klebsiella pneumoniae isolate harboring plasmid-mediated colistin resistance mcr-8 gene in Algeria.

OBJECTIVES: We aimed to develop here a specific real-time PCR assay with TaqMan® probe to detect efficiently bacterial strains harboring the new plasmid mediated-colistin resistance mcr-8 gene. METHODS: Specific primers and probe for mcr-8 gene were designed from sequences alignment of all mcr genes variants. Specificity of the designed primers and probe were first checked par BlastN analysis and by in silico PCR. The analytical sensitivity and specificity tests were performed in vitro on a panel of 290 genomic DNA of Gram-negative bacteria and 250 metagenomic DNA from human stool samples. Whole genome sequencing (WGS) was performed here using MiSeq technology. RESULTS: Designed primers and probe were 100% specific tomcr-8 gene by BlastN and in silico PCR analysis. Real-time PCR screening of a collection of clinical isolates resulted to one positive Klebsiella pneumoniae isolate (KP95). WGS confirmed that this isolate harbored the mcr-8 gene and other resistance genes such as blaOXA-48, blaCTX-M-15 ß-lactamases. Our real-time PCR was highly sensitive on a 10-fold dilution serie from a calibrated inoculum at 108 CFU/mL with a limit of detection at 55 CFU/mL. CONCLUSION: To the best of our knowledge, we propose here, the first real-time PCR assay targeting mcr-8 gene with high specificity and sensitivity, able to detect mcr-8 gene in less than 2 h from any DNA sample. This real-time PCR assay allowed the first description of a clinical K. pneumoniae strain harboring the mcr-8 gene in Algeria.

High Prevalence of Multidrug-Resistant Escherichia coli in Urine Samples from Inpatients and Outpatients at a Tertiary Care Hospital in Sétif, Algeria.

The worldwide dissemination of multidrug-resistant (MDR) Enterobacteriaceae is a major public health issue. The aim of this study was to investigate the prevalence of MDR Escherichia coli (MDR-EC) isolates, in inpatients/outpatients with urinary tract infections at Sétif University Hospital (Algeria). Bacterial cultures were obtained from 426 of the 3,944 urine samples collected from January 2015 to February 2017. Among these cultures, 215 E. coli isolates were identified by mass spectrometry, and 38 (17.7%) were MDR-EC (disk diffusion method): 36 produced only extended-spectrum ß-lactamases (ESBL), one ESBL and a carbapenemase, and one only a cephalosporinase (double-disk synergy test). Multiplex PCR and sequencing analyses showed that 37 ESBL-producing isolates harbored genes encoding CTX-M enzymes (CTX-M-15 in 33 isolates, 89.19%; and CTX-M-14 group in four isolates, 10.81%). One CTX-M-15-producing isolate co-expressed also an OXA-48-like carbapenemase. Phylogenetic group analysis of the 37 ESBL-producing and 178 non-ESBL-producing isolates indicated that the most common phylogenetic group was B2 (54.05% of ESBL-producing and 48.31% of non-ESBL-producing isolates), followed by A and D for ESBL-, and by B1, A, and F for non-ESBL-producing isolates. This is the first report highlighting the presence of MDR-EC isolates that produce both CTX-M and OXA-48-like enzymes in Sétif, Algeria.