Cost-effective in-house COVID-19 reverse transcription-polymerase chain reaction testing with yeast-derived Taq polymerase.

BACKGROUND: Despite the decline of the COVID-19 pandemic, there continues to be a persistent requirement for reliable testing methods that can be adapted to future outbreaks and areas with limited resources. While the standard approach of using reverse transcription-polymerase chain reaction (RT-PCR) with Taq polymerase is effective, it faces challenges such as limited access to high-quality enzymes and the presence of bacterial DNA contamination in commercial kits, which can impact the accuracy of test results. METHODS: This study investigates the production of recombinant Taq polymerase in yeast cells and assesses its crude lysate in a multiplex RT-PCR assay for detecting the SARS-CoV-2 RNA-dependent RNA polymerase (RdRP) and N genes, with human Ribonuclease P serving as an internal control. RESULTS: The unpurified yeast Taq polymerase demonstrates sensitivity comparable to commercially purified bacterial Taq polymerase and unpurified bacterial counterparts in detecting the RdRP and N genes. It exhibits the highest specificity, with 100% accuracy, for the N gene. The specificity for the RdRP gene closely aligns with that of commercially purified bacterial Taq polymerase and unpurified bacterial Taq polymerase. CONCLUSIONS: The use of unpurified recombinant yeast Taq polymerase shows promise as a cost-effective approach for conducting in-house COVID-19 RT-PCR testing. By eliminating the need for chromatography purification steps, the production of RT-PCR kits can be streamlined, potentially improving accessibility and scalability, especially in resource-limited settings and future pandemics.

Genomic insights into the diversity, virulence, and antimicrobial resistance of group B Streptococcus clinical isolates from Saudi Arabia.

Introduction: Detailed assessment of the population structure of group B Streptococcus (GBS) among adults is still lacking in Saudi Arabia. Here we characterized a representative collection of isolates from colonized and infected adults. Methods: GBS isolates (n=89) were sequenced by Illumina and screened for virulence and antimicrobial resistance determinants. Genetic diversity was assessed by single nucleotide polymorphisms and core-genome MLST analyses. Results: Genome sequences revealed 28 sequence types (STs) and nine distinct serotypes, including uncommon serotypes VII and VIII. Majority of these STs (n=76) belonged to the human-associated clonal complexes (CCs) CC1 (33.71%), CC19 (25.84%), CC17 (11.24%), CC10/CC12 (7.87%), and CC452 (6.74%). Major CCs exhibited intra-lineage serotype diversity, except for the hypervirulent CC17, which exclusively expressed serotype III. Virulence profiling revealed that nearly all isolates (94.38%) carried at least one of the four alpha family protein genes (i.e., alphaC, alp1, alp2/3, and rib), and 92.13% expressed one of the two serine-rich repeat surface proteins Srr1 or Srr2. In addition, most isolates harbored the pilus island (PI)-2a alone (15.73%) or in combination with PI-1 (62.92%), and those carrying PI-2b alone (10.11%) belonged to CC17. Phylogenetic analysis grouped the sequenced isolates according to CCs and further subdivided them along with their serotypes. Overall, isolates across all CC1 phylogenetic clusters expressed Srr1 and carried the PI-1 and PI-2a loci, but differed in genes encoding the alpha-like proteins. CC19 clusters were dominated by the III/rib/srr1/PI-1+PI-2a (43.48%, 10/23) and V/alp1/srr1/PI-1+PI-2a (34.78%, 8/23) lineages, whereas most CC17 isolates (90%, 9/10) had the same III/rib/srr2/P1-2b genetic background. Interestingly, genes encoding the CC17-specific adhesins HvgA and Srr2 were detected in phylogenetically distant isolates belonging to ST1212, suggesting that other highly virulent strains might be circulating within the species. Resistance to macrolides and/or lincosamides across all major CCs (n=48) was associated with the acquisition of erm(B) (62.5%, 30/48), erm(A) (27.1%, 13/48), lsa(C) (8.3%, 4/48), and mef(A) (2.1%, 1/48) genes, whereas resistance to tetracycline was mainly mediated by presence of tet(M) (64.18%, 43/67) and tet(O) (20.9%, 14/67) alone or in combination (13.43%, 9/67). Discussion: These findings underscore the necessity for more rigorous characterization of GBS isolates causing infections.

Molecular Basis of Antimicrobial Resistance in Group B Streptococcus Clinical Isolates from Saudi Arabia.

Published data on the molecular mechanisms underlying antimicrobial resistance in Group B Streptococcus (GBS) isolates from Saudi Arabia are lacking. Here, we aimed to determine the genetic basis of resistance to relevant antibiotics in a collection of GBS clinical isolates (n = 204) recovered from colonized adults or infected patients and expressing serotypes Ia, Ib, II, III, V, and VI. Initial susceptibility testing revealed resistance to tetracycline (76.47%, n = 156/204), erythromycin (36.76%, n = 75/204), clindamycin (25.49%, n = 52/204), levofloxacin (6.37%, n = 13/204), and gentamicin (2.45%, n = 5/204). Primers designed for the detection of known resistance determinants in GBS identified the presence of erm(A), erm(B), mef(A), and/or lsa(C) genes at the origin of resistance to macrolides and/or clindamycin. Of these, erm(B) and erm(A) were associated with the cMLSB (n = 46) and iMLSB (n = 28) phenotypes, respectively, while mef(A) was linked to the M phenotype (n = 1) and lsa(C) was present in isolates with the L phenotype (n = 8). Resistance to tetracycline was mainly mediated by tet(M) alone (n = 112) or in combination with tet(O) (n = 10); the remaining isolates carried tet(O) (n = 29), tet(L) (n = 2), or both (n = 3). Isolates resistant to gentamicin (n = 5) carried aac(6')-Ie-aph(2')-Ia, and those exhibiting resistance to levofloxacin (n = 13) had alterations in GyrA and/or ParC. Most isolates with the erm gene (93.24%, n = 69/74) also had the tet gene and were therefore resistant to erythromycin, clindamycin, and tetracycline. Overall, there were no clear associations between serotypes and resistance genotypes except for the presence of erm(B) in serotype Ib isolates. Dissemination of antibiotic resistance genes across different serotypes represents a public health concern that requires further surveillance and appropriate antibiotic use in clinical practice.

In vitro activity of cefiderocol against clinically important carbapenem non-susceptible Gram-negative bacteria from Saudi Arabia.

OBJECTIVES: Cefiderocol is a novel catechol-substituted siderophore cephalosporin with broad-spectrum activity against Gram-negative pathogens. However, variation of its activity among carbapenemase producers from various regions and countries has been reported. Here, we checked the in vitro activity against Gram-negative carbapenem non-susceptible bacteria collected in Saudi Arabia. METHODS: Cefiderocol MICs were determined using the iron-depleted cation-adjusted Mueller-Hinton broth and interpreted according to the Clinical and Laboratory Standards Institute guidelines. Isolates (n = 288) included carbapenemase-producing Escherichia coli (n = 46), Klebsiella pneumoniae (n = 98), Acinetobacter baumannii (n = 65), and Pseudomonas aeruginosa (n = 79) clinical isolates. RESULTS: Cefiderocol inhibited 73.26% (211/288) of the isolates studied at concentrations of ≤ 4 mg/L. Cefiderocol inhibited all carbapenem-resistant A. baumannii isolates (65/65, 100%) producing OXA-23-like, OXA-24-like, and NDM, and nearly all P. aeruginosa isolates (75/79, 94.94%), including those producing VIM and NDM. In contrast, the carbapenemase-producing isolates from the Enterobacterales group demonstrated significantly higher MICs with only 53.06% (52/98) of K. pneumoniae and 41.3% (19/46) of E. coli isolates exhibiting MICs of ≤4 mg/L. Isolates showing elevated MICs (73/144, 50.69%) included NDM (20/29, 68.97%), NDM/OXA-48-like (34/59, 57.63%), OXA-48-like (18/52, 34.62%), and KPC (1/4, 25%) producers, thus showing no clear association with the production of serine-type or metallo-type carbapenemases. However, high cefiderocol MICs (≥ 32mg/L) were associated with isolates producing NDM, and in particular, among those coproducing the OXA-232-type enzyme. CONCLUSIONS: Cefiderocol had excellent activity against multi-drug resistant non-fermenting Gram-negative pathogens. Reasons behind the high cefiderocol MICs in certain Enterobacterales isolates need further investigation.

Molecular typing and antimicrobial resistance of group B Streptococcus clinical isolates in Saudi Arabia.

OBJECTIVES: Group B Streptococcus (GBS) has emerged as an important cause of severe infections in adults. However, limited data are available regarding the epidemiology of GBS in Saudi Arabia. METHODS: Isolates were collected over a period of eight months from colonized (n = 104) and infected adults (n = 95). Serotypes and virulence determinants were detected by polymerase chain reactions (PCRs). Genetic relatedness was assessed using Multiple Locus Variable Number Tandem Repeat Analysis (MLVA). Antimicrobial susceptibilities were determined by disk diffusion. RESULTS: Serotypes III and V (25% each) were the most prevalent, followed by serotypes II (16.18%), Ia (13.24%), VI (9.31%), and Ib (8.82%), while five isolates remained non-typeable (2.45%). Hypervirulent serotype III/CC17 clone (n = 21) accounted for 41.18% of the serotype III isolates. Most isolates (53.92%) harboured pilus island (PI) 1 and 2a types, while PI-2b was predominantly detected in the hypervirulent clone. Isolates were variably resistant to tetracycline (76.47%), erythromycin (36.76%), clindamycin (25.49%), and levofloxacin (6.37%), but remained susceptible to penicillin. Macrolide resistant isolates exhibited constitutive (55.42%) and inducible macrolide-lincosamide-streptogramin B resistance phenotypes (33.74%), while a few had L (9.64%) or M (1.2%) phenotypes. MLVA patterns of dominant serotypes III and V revealed 40 different types divided into 12 clusters and 28 singletons. Interestingly, macrolide resistance was significantly associated with two major MLVA types. CONCLUSIONS: GBS isolates belonged predominantly to serotypes III and V, but there were no clear associations between serotypes and patient groups. The studied isolates exhibited high levels of resistance to erythromycin and clindamycin that need further surveillance.

Epididymo-Orchitis Caused by POM-1 Metallo-ß-Lactamase-Producing Pseudomonas otitidis in an Immunocompetent Patient: Case Report and Molecular Characterization.

Pseudomonas otitidis is a rare and unique species among the Pseudomonas genus that has not been previously reported as a cause of male genitourinary tract infection. In this report, we describe a case of a 20-year-old immunocompetent male who presented with recurrent epididymo-orchitis, which was initially misidentified as Vibrio vulnificus and treated successfully. The causative agent could not be identified appropriately using the available routine methods, but a final identification was established using 16S rRNA targeted sequencing followed by whole-genome sequencing.

Genomic Characterization of Carbapenem-Non-susceptible Pseudomonas aeruginosa Clinical Isolates From Saudi Arabia Revealed a Global Dissemination of GES-5-Producing ST235 and VIM-2-Producing ST233 Sub-Lineages.

Carbapenem-resistant P. aeruginosa has become a major clinical problem due to limited treatment options. However, studies assessing the trends in the molecular epidemiology and mechanisms of antibiotic resistance in this pathogen are lacking in Saudi Arabia. Here, we reported the genome characterization in a global context of carbapenem non-susceptible clinical isolates from a nationally representative survey. The antibiotic resistance profiles of the isolates (n = 635) collected over 14 months between March 2018 and April 2019 from different geographical regions of Saudi Arabia showed resistance rates to relevant ß-lactams, aminoglycosides and quinolones ranging between 6.93 and 27.56%. Overall, 22.52% (143/635) of the isolates exhibited resistance to both imipenem and meropenem that were mainly explained by porin loss and efflux overexpression. However, 18.18% of resistant isolates harbored genes encoding GES (69.23%), VIM (23.07%), NDM (3.85%) or OXA-48-like (3.85%) carbapenemases. Most common GES-positive isolates produced GESs -5, -15 or -1 and all belonged to ST235 whereas the VIM-positive isolates produced mainly VIM-2 and belonged to ST233 or ST257. GES and VIM producers were detected at different sampling periods and in different surveyed regions. Interestingly, a genome-wide comparison revealed that the GES-positive ST235 and VIM-2-positive ST233 genomes sequenced in this study and those available through public databases from various locations worldwide, constituted each a phylogenetically closely related sub-lineage. Profiles of virulence determinants, antimicrobial resistance genes and associated mobile elements confirmed relatedness within each of these two different sub-lineages. Sequence analysis located the bla GES gene in nearly all studied genomes (95.4%) in the same integrative conjugative element that also harbored the acc(6')-Ib, aph(3')-XV, aadA6, sul1, tet(G), and catB resistance genes while bla VIM-2 in most (98.89%) ST233-positive genomes was co-located with aac(6')-I1, dfrB-5, and aac(3')-Id in the same class I integron. The study findings revealed the global spread of GES-5 ST235 and VIM-2 ST233 sub-lineages and highlighted the importance of routine detection of rare ß-lactamases.

Invasive fungal infection of the brain caused by Neoscytalidium dimidiatum in a post-renal transplant patient: A case report.

Neoscytalidium is a phytopathogen that is often found in plants and soil. It mostly leads to skin and nail infections, and invasive diseases of the sinuses, lung, and brain have been described mostly in immunocompromised patients. We report a case of a post-renal transplant patient who received anti-thymocyte globulin for induction immunosuppression. A month after her transplant, she presented with fever and new-onset seizures, and computed tomography revealed a brain abscess with mass effects and herniation. The patient underwent abscess drainage and craniectomy. The pathological findings showed filamentous septate hyphae. The surgical culture rapidly grew wool-like colonies with a black reverse on Sabouraud agar. Lactophenol cotton blue staining showing septate branched hyphae with one to two arthroconidia cells with flattened ends. The patient was given a combination of amphotericin B and voriconazole but unfortunately died ten days after the diagnosis. This case highlights Neoscytalidium as a cause of invasive fungal disease in immunocompromised patients that is difficult to treat and is often fatal, even when combined surgical and medical therapies are used as treatment modalities.

Successful treatment of infective endocarditis due to pandrug-resistant Klebsiella pneumoniae with ceftazidime-avibactam and aztreonam.

Pandrug-resistant (PDR) K. pneumoniae refractory to conventional treatment has been reported worldwide, causing a huge burden on the healthcare system, patient safety and the economy. K. pneumoniae is a prominent opportunistic pathogen causing hospital-acquired and community-acquired infections, but is rarely associated with infective endocarditis. Currently, there are sparse data guiding the optimal regimen when commonly used antibiotics fail, notably for the treatment of endocarditis infections. Here we report our experience in treating a 40-year-old female with PDR K. pneumoniae infection of cardiovascular implantable electronic device (CIED) and right-sided infective endocarditis. Initial susceptibility testing of the incriminated pathogen showed an apparent susceptibility to colistin but the prolonged course of colistin, gentamicin and meropenem did not resolve the infection. However, the synergistic combinations of aztreonam with ceftazidime-avibactam was able to overcome resistance and clear the infection rapidly. Genome sequencing showed that the PDR K. pneumoniae isolate belongs to the international high-risk clone ST14. The isolate harbored genes encoding NDM-1, OXA-48, CTX-M-14b, SHV-28 and OXA-1, explaining resistance to all ß-lactams, including carbapenems. It carried the armA gene conferring resistance to all clinically important aminoglycosides and had alterations in GyrA, ParC and MgrB, explaining resistance to ciprofloxacin and colistin.

Complete Genome Sequence of a Colistin-Susceptible Salmonella enterica Serovar Minnesota Strain Harboring mcr-9 on an IncHI2/IncHI2A Plasmid, Isolated from Chicken Meat.

The recent emergence and dissemination of mobilized colistin resistance (mcr) genes have triggered extensive concerns globally. Here, we report the complete genome sequence of a colistin-susceptible Salmonella enterica serotype Minnesota strain (named SA18578), belonging to sequence type 548 (ST548) and carrying the mcr-9 gene on an IncHI2/IncHI2A plasmid, that was isolated from chicken meat in Saudi Arabia in 2020.

Complete Genome Sequence of a Colistin-Resistant Uropathogenic Escherichia coli Sequence Type 131 fimH22 Strain Harboring mcr-1 on an IncHI2 Plasmid, Isolated in Riyadh, Saudi Arabia.

We report the complete genome sequence of a colistin-resistant strain of uropathogenic Escherichia coli, isolated in January 2013 at King Abdulaziz Medical City (KAMC), Riyadh, Saudi Arabia. The isolate (named SA186) was sequence type 131 (ST131) and belonged to serotype O25b-H4 and clade B (fimH22).

Occurrence of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) across the Gulf Corporation Council countries: Four years update.

The emergence of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infections has become a global issue of dire concerns. MERS-CoV infections have been identified in many countries all over the world whereas high level occurrences have been documented in the Middle East and Korea. MERS-CoV is mainly spreading across the geographical region of the Middle East, especially in the Arabian Peninsula, while some imported sporadic cases were reported from the Europe, North America, Africa, and lately Asia. The prevalence of MERS-CoV infections across the Gulf Corporation Council (GCC) countries still remains unclear. Therefore, the objective of the current study was to report the prevalence of MERS-CoV in the GCC countries and to also elucidate on its demographics in the Arabian Peninsula. To date, the World Health Organization (WHO) has reported 1,797 laboratory-confirmed cases of MERS-CoV infection since June 2012, involving 687 deaths in 27 different countries worldwide. Within a time span of 4 years from June 2012 to July 2016, we collect samples form MERS-CoV infected individuals from National Guard Hospital, Riyadh, and Ministry of health Saudi Arabia and other GCC countries. Our data comprise a total of 1550 cases (67.1% male and 32.9% female). The age-specific prevalence and distribution of MERS-CoV was as follow: <20 yrs (36 cases: 3.28%), 20-39 yrs (331 cases: 30.15%), 40-59 yrs (314 cases: 28.60%), and the highest-risk elderly group aged ≥60 yrs (417 cases: 37.98%). The case distribution among GCC countries was as follows: Saudi Arabia (1441 cases: 93%), Kuwait (4 cases: 0.3%), Bahrain (1 case: 0.1%), Oman (8 cases: 0.5%), Qatar (16 cases: 1.0%), and United Arab Emirates (80 cases: 5.2%). Thus, MERS-CoV was found to be more prevalent in Saudi Arabia especially in Riyadh, where 756 cases (52.4%) were the worst hit area of the country identified, followed by the western region Makkah where 298 cases (20.6%) were recorded. This prevalence update indicates that the Arabian Peninsula, particularly Saudi Arabia, is the hardest hit region regarding the emerging MERS-CoV infections worldwide. GCC countries including Saudi Arabia now have the infrastructure in place that allows physicians and scientific community to identify and immediately respond to the potential risks posed by new outbreaks of MERS-CoV infections in the region. Given the continuum of emergence and the large magnitude of the disease in our region, more studies will be required to bolster capabilities for timely detection and effective control and prevention of MERS-CoV in our region.