Co-Occurrence of ß-Lactam and Aminoglycoside Resistance Determinants among Clinical and Environmental Isolates of Klebsiella pneumoniae and Escherichia coli: A Genomic Approach.

The presence of antimicrobial-resistance genes (ARGs) in mobile genetic elements (MGEs) facilitates the rapid development and dissemination of multidrug-resistant bacteria, which represents a serious problem for human health. This is a One Health study which aims to investigate the co-occurrence of antimicrobial resistance determinants among clinical and environmental isolates of K. pneumoniae and E. coli. Various bioinformatics tools were used to elucidate the bacterial strains' ID, resistome, virulome, MGEs, and phylogeny for 42 isolates obtained from hospitalized patients (n = 20) and environmental sites (including fresh vegetables, fruits, and drinking water) (n = 22). The multilocus sequence typing (MLST) showed that K. pneumoniae belonged to ten sequence types (STs) while the E. coli belonged to seventeen STs. Multidrug-resistant isolates harbored ß-lactam, aminoglycoside resistance determinants, and MGE were detected circulating in the environment (drinking water, fresh vegetables, and fruits) and in patients hospitalized with postoperative infections, neonatal sepsis, and urinary tract infection. Four K. pneumoniae environmental isolates (7E, 16EE, 1KE, and 19KE) were multidrug-resistant and were positive for different beta-lactam and aminoglycoside resistance determinants. blaCTX-M-15 in brackets of ISEc 9 and Tn 3 transposases was detected in isolates circulating in the pediatrics unit of Soba hospital and the environment. This study documented the presence of bacterial isolates harboring a similar pattern of antimicrobial resistance determinants circulating in hospitals and environments. A rapid response is needed from stakeholders to initiate a program for infection prevention and control measures to detect such clones disseminated in the communities and hospitals.

Whole-Genome Sequence of Multidrug-Resistant Methicillin-Resistant Staphylococcusepidermidis Carrying Biofilm-Associated Genes and a Unique Composite of SCCmec.

Staphylococcus epidermidis is part of the normal human flora that has recently become an important opportunistic pathogen causing nosocomial infections and tends to be multidrug-resistant. In this investigation, we aimed to study the genomic characteristics of methicillin-resistant S. epidermidis isolated from clinical specimens. Three isolates were identified using biochemical tests and evaluated for drug susceptibility. Genomic DNA sequences were obtained using Illumina, and were processed for analysis using various bioinformatics tools. The isolates showed multidrug resistance to most of the antibiotics tested in this study, and were identified with three types (III(3A), IV(2B&5), and VI(4B)) of the mobile genetic element SCCmec that carries the methicillin resistance gene (mecA) and its regulators (mecI and mecR1). A total of 11 antimicrobial resistance genes (ARGs) was identified as chromosomally mediated or in plasmids; these genes encode for proteins causing decreased susceptibility to methicillin (mecA), penicillin (blaZ), fusidic acid (fusB), fosfomycin (fosB), tetracycline (tet(K)), aminoglycosides (aadD, aac(6')-aph(2'')), fluoroquinolone (MFS antibiotic efflux pump), trimethoprim (dfrG), macrolide (msr(A)), and chlorhexidine (qacA)). Additionally, the 9SE strain belongs to the globally disseminated ST2, and harbors biofilm-formation genes (icaA, icaB, icaC, icaD, and IS256) with phenotypic biofilm production capability. It also harbors the fusidic acid resistance gene (fusB), which could increase the risk of device-associated healthcare infections, and 9SE has been identified as having a unique extra SCC gene (ccrB4); this new composite element of the ccr type needs more focus to better understand its role in the drug resistance mechanism.

Genomic Analysis of Multidrug-Resistant Hypervirulent (Hypermucoviscous) Klebsiella pneumoniae Strain Lacking the Hypermucoviscous Regulators (rmpA/rmpA2).

Hypervirulent K. pneumoniae (hvKP) strains possess distinct characteristics such as hypermucoviscosity, unique serotypes, and virulence factors associated with high pathogenicity. To better understand the genomic characteristics and virulence profile of the isolated hvKP strain, genomic data were compared to the genomes of the hypervirulent and typical K. pneumoniae strains. The K. pneumoniae strain was isolated from a patient with a recurrent urinary tract infection, and then the string test was used for the detection of the hypermucoviscosity phenotype. Whole-genome sequencing was conducted using Illumina, and bioinformatics analysis was performed for the prediction of the isolate resistome, virulome, and phylogenetic analysis. The isolate was identified as hypermucoviscous, type 2 (K2) capsular polysaccharide, ST14, and multidrug-resistant (MDR), showing resistance to ciprofloxacin, ceftazidime, cefotaxime, trimethoprim-sulfamethoxazole, cephalexin, and nitrofurantoin. The isolate possessed four antimicrobial resistance plasmids (pKPN3-307_type B, pECW602, pMDR, and p3K157) that carried antimicrobial resistance genes (ARGs) (blaOXA-1,blaCTX-M-15, sul2, APH(3″)-Ib, APH(6)-Id, and AAC(6')-Ib-cr6). Moreover, two chromosomally mediated ARGs (fosA6 and SHV-28) were identified. Virulome prediction revealed the presence of 19 fimbrial proteins, one aerobactin (iutA) and two salmochelin (iroE and iroN). Four secretion systems (T6SS-I (13), T6SS-II (9), T6SS-III (12), and Sci-I T6SS (1)) were identified. Interestingly, the isolate lacked the known hypermucoviscous regulators (rmpA/rmpA2) but showed the presence of other RcsAB capsule regulators (rcsA and rcsB). This study documented the presence of a rare MDR hvKP with hypermucoviscous regulators and lacking the common capsule regulators, which needs more focus to highlight their epidemiological role.

Molecular insights into novel environmental strains of Klebsiella quasipneumoniae harboring different antimicrobial-resistance genes.

Introduction: The emergence of bacterial pathogens in environmental hosts represents a major risk to public health. This study aimed at characterizing seven novel environmental strains of K. quasipneumoniae using a genomic approach which was misidentified by phenotypic methods in a previous batch of 27 species thought to be K. pneumoniae. Methods: Whole-genome sequencing was performed using the Illumina platform, and the generated raw reads were de novo assembled. Comparative genomic, resistome, virulome, mobilome, and phylogeny were then investigated using dierent bioinformatics tools. Results: Six strains were identified as K. quasipneumoniae subsp similipneumoniae and one as K. quasipneumoniae subsp. quasipneumoniae. All isolates were resistant to ampicillin, cephalexin, and amoxicillin-clavulanic acid and harbored the fosA, bla OKP types, oqxB, and oqxA genes. One isolate additionally harbored a gene cassettes consisting of bla SHV-1, bla OXA-1, aac(6')-Ib-cr, catB genes. The aminoglycoside-modifying enzyme gene aph(3")-Ia was bracketed by two insertion elements. Plasmid analyses showed that IncFIBK was the most prevalent plasmid, circulating in six isolates, while one isolate exhibited seven different plasmids. The isolates have virulence genes responsible for capsule formation, lipopolysaccharide, iron uptake aerobactin (iutA), salmochelins (iroE, iroN), enterobactin siderophore, adherence, and biofilm formation (mrkA, mrkB, mrkC, mrkD, mrkF, and mrkH). Conclusion: Our study highlights the ecology and transmission of K. quasipneumoniae (which have the ability to disseminate to other environmental sources including animals) outside the clinical setting and the contribution of water, vegetables, and table surfaces as potential reservoirs of farm-to-fork transmission of disease via local markets in Khartoum, Sudan.

Detection and characterization of carbapenem resistant Gram-negative bacilli isolates recovered from hospitalized patients at Soba University Hospital, Sudan.

BACKGROUND: Antimicrobial resistance (AMR) poses a complex threat to global health security and universal health coverage. Recently, nosocomial infections with carbapenemase-producing Gram-negative bacilli (GNB) is increasing worldwide. We report the molecular characterization and detection of genes associated with carbapenemase producing Gram negative bacteria isolated from hospitalized patients at Soba University Hospital (SUH) in Khartoum State, Sudan. RESULTS: Between October 2016 and February 2017, a total of 206 GNB clinical specimens were collected from hospitalized patients in SUH. Of 206 carbapenem resistance isolates, 171 (83 %) were confirmed as phenotypically resistant and 121 (58.7 %) isolates harboured one or more carbapenemase genes. New Delhi metallo-ß-lactamase (NDM) types were the most predominant genes, blaNDM 107(52 %), followed by blaIMP 7 (3.4 %), blaOXA-48 5(2.4 %) and blaVIM 2 (0.9 %). Co-resistance genes with NDM producing GNB were detected in 87 (81.3 %) of all blaNDM producing isolates. NDM-1 was the most frequent subtype observed in 75 (70 %) blaNDM producing isolates. The highest percentage of resistance was recorded in ampicillin (98 %), cephalexin (93.5 %) amoxicillin clavulanic acid (90 %), cefotaxime (89.7 %), ceftriaxone (88.4 %), ceftazidime (84.2 %), sulfamethoxazole-trimethoprim (78.4 %) and nitrofurantoin (75.2 %), aztreonam (66 %) and temocillin (64 %). A close correlation between phenotypic and carbapenemase genes detection in all GNB was observed. CONCLUSIONS: The frequency of carbapenemase producing bacilli was found to be high in SUH. NDM was found to be the most prevalent carbapenemase gene among clinical isolates. Close surveillance across all hospitals in Sudan is required. The relative distribution of carbapenemase genes among GNB in nosocomial infections in Africa needs to be defined.

Use of immunoblotting in testing Madurella mycetomatis specific antigen.

BACKGROUND: Though serodiagnosis of actinomycetoma is established, that of eumycetoma due to Madurella mycetomatis is limited because of lack of pure antigen. Reliable rapid tests are needed to make an accurate timely diagnosis. The purpose of this study is to detect antigen parts of M. mycetomatis, which act specifically with M. mycetomatis antibodies. METHODS: Cytoplasmic antigen was prepared from molecularly identified cultures of M. mycetomatis by sonication, ultracentrifugation, dried, weighed and appropriately reconstituted. M. mycetomatis cytoplasmic antigen were separated using 12% sodium dodecyl sulfate-polyacrylamide gel, and immunoblotting to detect the reactive ones.Immunoblotting was carried out in nitrocellulose strips containing different molecular size. Sera from patients and co-patients as control were used. RESULTS: When stained with Coomassie brilliant blue R 250 seven molecular weights appeared but only three, 45, 60, 95 kDa reacted with M. mycetomatis patients few from control group, one from a malaria patient. No reactive band was observed with sera from actinomycetoma, Aspergillus flavus-associated aspergillosis, schistosomiasis, leishmaniasis, fungal sinusitis nor healthy controls. CONCLUSIONS: Specific fractions of M. mycetomatis antigen which were demonstrated by immunoblotting showed 75% sensitivity and 95% specificity. The true negative tests were 14 patients (32.5%). This also means that immunoblotting is reasonably reliable in diagnosis and follow-up of eumycetoma patients.