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 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.

Molecular characterization of clinical and environmental carbapenem resistant Acinetobacter baumannii isolates in a hospital of the Eastern Region of Saudi Arabia.

BACKGROUND: Environmental and clinical carbapenem-resistant Acinetobacter baumannii (CRAb) isolated in a hospital of the Eastern Region of Saudi Arabia were compared to assess the potential environmental contamination by this pathogen. METHODS: Frequent-hand-touch surfaces of intensive care (ICU), medical (MW), and surgical (SW) units were randomly sampled for a month-long period, and the CRAb identified were compared to clinical isolates of the same period by PFGE and blaOXA-51-like gene sequencing. Carbapenemase and ribosomal methylase genes, ISAba1 link to blaOXA51-like or to blaOXA-23, respectively were detected by PCR. RESULTS: CRAb was identified from 35.5% of surfaces. All environmental and clinical isolates were multi- or extremely drug resistant. PFGE of all clinical (n=21) and selected environmental (n=30) isolates identified a singleton and four clusters, all of which included both clinical and environmental isolates. In the two largest clusters isolates carried blaOXA-66, ISAba1-linked blaOXA-23, and were from the ICU, MW and the male SW. Isolates of the female SW carried blaOXA-69, ISAba1-linked blaOXA-23 and blaGES-11. A pair of clinical and environmental CRAb from the Male SW harboured blaNDM-1 in addition to ISAba1-linked blaOXA-94. CONCLUSION: A worrying level of environmental contamination, often by CRAb belonging to international clones, was revealed, highlighting the importance of environmental hygiene.

Incidence and antibiotic susceptibility of MRSA infections in a Saudi Arabian Hospital: a 10-year surveillance study.

INTRODUCTION: Methicillin-resistant Staphylococcus aureus (MRSA) infections remain prevalent and are associated with significant morbidity and mortality. The aim of the present study was to investigate the epidemiology of MRSA infections and antibiotic susceptibility in Qatif, Saudi Arabia. METHODOLOGY: All patients who had positive culture for S. aureus from January 1, 2006 through December 31, 2015 were enrolled and analyzed in WHONET, a free database software developed by the World Health Organization (WHO). Patients' data were collected from electronic medical records and traditional chart reviews to determine whether MRSA acquisition was likely to have been in the community or in the healthcare facility. Susceptibility results for community-associated (CA)-MRSA were compared with isolates from healthcare setting. RESULTS: A total of 3395 patients with S. aureus infections were analyzed, with an overall annual MRSA incidence of 25 cases per 100,000 patients (27% of total S. aureus isolates). While the majority (64%) of MRSA infections occurred in healthcare setting, CA-MRSA isolation increased steadily from 23% in 2006 to 60% in 2015, exceeding rate of isolation of healthcare-associated (HA)-MRSA. Skin and soft tissue, the lung and blood stream were the most common sites of infection, with 20% to 35% of MRSA infections occurring in pediatric patients. In the inpatient setting, the majority of infections due to MRSA were in surgical wards and critical care units. Compared with CA-MRSA, HA-MRSA isolates turned out to be more frequently resistant against ciprofloxacin, clindamycin, erythromycin, tetracycline, and trimethoprim/sulfamethoxazole. CONCLUSIONS: Staphylococcus aureus continues to cause multiple site infections with a relatively stable methicillin-resistance rate, but the isolation of MRSA from the community is increasing.

Nurses' competency in drawing blood cultures and educational intervention to reduce the contamination rate.

Compared with truly negative cultures, false positive blood cultures (BCs) not only increase laboratory work but also prolong the lengths of patient stays, which are likely to increase patient morbidity and costs. The present study aimed to evaluate the effectiveness of a hospital-wide educational intervention on BC contamination rates. Nurses performed all phlebotomies; therefore, educational workshops were offered to all nurses twice a week over a 3-month period. The workshops consisted of a questionnaire, PowerPoint presentation, video show, demonstration of the different materials used to collect BCs, and question session. Data from the questionnaires and laboratory culture results were compared between the 6-month pre- and post-intervention periods. Of the 503 eligible nurses, 216 (42.9%) attended the workshops. The survey identified areas for improvement, which included time of disinfectant application, volume of blood to be cultured, and disinfection of BC bottle tops. Of the 9903 BC sets that were drawn from 3649 patients during the study period, 676 (6.8%) were contaminated. The monthly BC contamination rates for the 6-month pre- and post-intervention periods were 8.1% and 5.2%, respectively, representing a 36% reduction (P=0.008). Only three wards had an acceptable contamination rate of ≤3% before the intervention, compared with eight wards after the intervention. While contamination of BCs can never be completely eliminated, there is evidence that adherence to best practice BC collection techniques can minimize BC contamination, which might be best achieved with a dedicated phlebotomy team.

Enhancement of antibiotic susceptibility of Stenotrophomonas maltophilia using a polyclonal antibody developed against an ABC multidrug efflux pump.

Stenotrophomonas maltophilia is an emerging nosocomial pathogen capable of causing healthcare-associated infections, including pneumonia and bacteremia. Intrinsic resistance in S. maltophilia is exhibited towards many broad-spectrum antibiotics, and treatment recommendations are controversial. One of the major causes of antimicrobial resistance is attributed to a robust array of efflux pumps that extrude drug compounds from the cell. Using checkerboard and growth kinetic assays, we evaluated the in vitro activity of a polyclonal antibody raised against an ATP-binding cassette efflux protein in S. maltophilia. Six clinical strains of S. maltophilia and one type strain were challenged with co-trimoxazole, ticarcillin-clavulanate, and ciprofloxacin, alone and in combination with antibody. One clinical strain was tested by growth curve experiments for each antibiotic-antibody combination. The use of antibody resulted in significantly increased susceptibility in 71.4% (15/21) of treatments tested, with 33.3% displaying synergy and 38.1% an additive effect. In growth kinetic studies, synergy was obtained for each antibiotic-antibody combination. Thus, the use of antibody raised against multidrug efflux pumps for the treatment of multidrug-resistant organisms warrants further investigation. Antibody targeting substrate recognition sites, or other functionally important epitopes, may lead to inhibition of multiple efflux pumps that share the same substrate and is an attractive area that should be explored.

Molecular cloning and characterization of SmrA, a novel ABC multidrug efflux pump from Stenotrophomonas maltophilia.

OBJECTIVES: Stenotrophomonas maltophilia is an emerging nosocomial pathogen that can cause difficult-to-treat infections and exhibits significant degrees of poorly understood multidrug resistance (MDR). The aim of this study was to identify and characterize a multidrug ATP-binding cassette (ABC) efflux pump in S. maltophilia. METHODS: SmrA was identified in the S. maltophilia genome based on the detection of ABC transporter conserved motifs and alignment with experimentally proven MDR ABC transporters. The smrA gene was cloned and expressed in the hypersusceptible acrAB mutant Escherichia coli strain SM1411. The resistance to several antimicrobial agents was tested using Stokes' disc diffusion and broth microdilution MIC methods. Norfloxacin accumulation and efflux assays were performed using a fluorescence method with and without the efflux pump inhibitors sodium O-vanadate and reserpine. RESULTS: Cloning and expression of smrA in Escherichia coli conferred increased resistance to structurally unrelated compounds, including fluoroquinolones, tetracycline, doxorubicin and multiple dyes. Moreover, the expression of smrA in E. coli reduced norfloxacin uptake and enhanced its efflux, features that could be inhibited by the ABC efflux pump inhibitors. CONCLUSIONS: SmrA is a member of the ABC multidrug efflux pump family. The findings warrant further study of the role of this molecule in S. maltophilia isolates, to estimate the potential impact of this system in antimicrobial resistance.