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Phenotypic and genotypic characterization of clinical Staphylococcus aureus isolates from Kenya.

ABSTRACT: BACKGROUND:The increase and spread of virulent-outbreak associated, methicillin and vancomycin resistant (MRSA/VRSA) Staphylococcus aureus require a better understanding of the resistance and virulence patterns of circulating and emerging strains globally. This study sought to establish the resistance phenotype, and strains of 32 non-duplicate clinical MRSA and MSSA S. aureus isolates from four Kenyan hospitals, identify their resistance and virulence genes and determine the genetic relationships of MRSA with global strains. METHODS:Antimicrobial susceptibility profiles were determined on a Vitek 2, genomic DNA sequenced on an Illumina Miseq and isolates typed in-silico. Resistance and virulence genes were identified using ARIBA and phylogenies generated using RAxML. RESULTS:The MRSA isolates were 100% susceptible to vancomycin, teicoplanin, linezolid, and tigecycline. Nine distinct CC, 12 ST and 15 spa types including the novel t17826 and STs (4705, 4707) were identified with CC8 and CC152 predominating. MRSA isolates distributed across 3 CCs; CC5-ST39 (1), CC8 - ST241 (4), a novel CC8-ST4705 (1), ST8 (1) and CC152 (1). There was >?90% phenotype-genotype concordance with key resistance genes identified only among MRSA isolates: gyrA, rpoB, and parC mutations, mecA, ant (4')-lb, aph (3')-IIIa, ermA, sat-4, fusA, mphC and msrA. Kenyan MRSA isolates were genetically diverse and most closely related to Tanzanian and UK isolates. There was a significant correlation between map, hlgA, selk, selq and cap8d virulence genes and severe infections. CONCLUSION:The findings showed a heterogeneous S. aureus population with novel strain types. Though limited by the low number of isolates, this study begins to fill gaps and expand our knowledge of S. aureus epidemiology while uncovering interesting patterns of distribution of strain types which should be further explored. Although last-line treatments are still effective, the potential for outbreaks of both virulent and resistant strains remain, requiring sustained surveillance of S. aureus populations.

SUBMITTER: Kyany'a C

PROVIDER: S-EPMC6836327 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Phenotypic and genotypic characterization of clinical Staphylococcus aureus isolates from Kenya.

Kyany'a Cecilia C Nyasinga Justin J Matano Daniel D Oundo Valerie V Wacira Simon S Sang Willie W Musila Lillian L

BMC microbiology 20191106 1

<h4>Background</h4>The increase and spread of virulent-outbreak associated, methicillin and vancomycin resistant (MRSA/VRSA) Staphylococcus aureus require a better understanding of the resistance and virulence patterns of circulating and emerging strains globally. This study sought to establish the resistance phenotype, and strains of 32 non-duplicate clinical MRSA and MSSA S. aureus isolates from four Kenyan hospitals, identify their resistance and virulence genes and determine the genetic rela ...[more]

PMID: 31694531

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Project description:Staphylococcus aureus is a commensal and frequent colonizer of the upper respiratory tract. When mechanical ventilation disrupts natural defenses, S. aureus is frequently isolated from the lower airways, but distinguishing between colonization and infection is difficult. The objectives of this study were (1) to investigate the bacterial genome sequence in consecutive isolates in order to identify changes related to the pathological adaptation to the lower respiratory tract and (2) to explore the relationship between specific phenotypic and genotypic features with the patient's study group, persistence of the clinical isolate and clinical outcome. A set of 94 clinical isolates were selected and corresponded to 34 patients that were classified as having pneumonia (10), tracheobronchitis (11) and bronchial colonization (13). Clinical strains were phenotypically characterized by conventional identification and susceptibility testing methods. Isolates underwent whole genome sequencing using Illumina HiSeq4000. Genotypic characterization was performed with an in-house pipeline (BacterialTyper). Genomic variation arising within-host was determined by comparing mapped sequences and de novo assemblies. Virulence factors important in staphylococcal colonization and infection were characterized using previously established functional assays. (1) Toxin production was assessed using a THP-1 cytotoxicity assay, which reports on the gross cytotoxicity of individual isolates. In addition, we investigated the expression of the major virulence factor, alpha-toxin (Hla) by Western blot. (2) Adhesion to the important extracellular matrix molecule, fibronectin, was determined using a standardized microtitre plate assay. Finally, invasion experiments using THP-1 and A539 cell lines and selected clinical strains were also performed. Repeated isolation of S. aureus from endotracheal aspirate usually reflects persistence of the same strain. Within-host variation is detectable in this setting, but it shows no evidence of pathological adaptation related to virulence, resistance or niche adaptations. Cytotoxicity was variable among isolates with 14 strains showing no cytotoxicity, with these latter presenting an unaltered Fn binding capacity. No changes on cytotoxicity were reported when comparing study groups. Fn binding capacity was reported for almost all strains, with the exception of two strains that presented the lowest values. Strains isolated from patients with pneumonia presented a lower capacity of adhesion in comparison to those isolated during tracheobronchitis (p = 0.002). Hla was detected in 71 strains (75.5%), with most of the producer strains in pneumonia and bronchial colonization group (p = 0.06). In our cohort, Hla expression (presence or absence) in sequential isolates was usually preserved (70%) although in seven cases the expression varied over time. No relationship was found between low cytotoxicity and intracellular persistence in invasion experiments. In our study population, persistent S. aureus isolation from airways in ventilated patients does not reflect pathological adaptation. There is an important diversity of sequence types. Cytotoxicity is variable among strains, but no association with study groups was found, whereas isolates from patients with pneumonia had lower adhesion capability. Favorable clinical outcome correlated with increased bacterial adhesion in vitro. Most of the strains isolated from the lower airways were Hla producers and no correlation with an adverse outcome was reported. The identification of microbial factors that contribute to virulence is relevant to optimize patient management during lower respiratory tract infections.

Project description:Background & objectivesEnterococci have emerged as important nosocomial pathogens and emergence of resistance to many of the antimicrobials used for Gram-positive organisms has made the management of infections due to Enterococcus species difficult. Resistance to glycopeptide antibiotics, especially vancomycin is of special concern. This study was undertaken to perform a phenotypic and genotypic characterization of vancomycin resistant Enterococcus (VRE) isolates obtained from clinical samples in a tertiary care hospital in southern India.MethodsSusceptibility testing was performed for Enterococcus isolates collected over a period of one year (November 2008-October 2009). Minimum inhibitory concentrations (MIC) of vancomycin and teicoplanin were determined for the isolates by the agar dilution method. Genotypic characterization of VRE isolates was done by performing multiplex polymerase chain reaction (PCR) for detecting the various vancomycin resistance genes.ResultsOf the 367 isolates of Enterococcus species isolated, 32 were found to be resistant to vancomycin after MIC testing. VanA was the commonest phenotype of vancomycin resistance and the commonest genotype was vanA. Among the other important findings of the study was the presence of heterogeneity in isolates of VRE with the vanA gene cluster with regards to resistance to teicoplanin and the coexistence of vanA and vanC1 gene clusters in an isolate of E. gallinarum which conferred high level glycopeptide resistance to the isolate.Interpretation & conclusionsEnterococcus species have emerged as important nosocomial pathogens in our patients with a capacity to cause a variety of infections. The vancomycin resistance among Enterococcus isolates was 8.7 per cent in our study which was high compared to other Indian studies. VanA was the commonest phenotype of glycopeptide resistance and vanA was the commonest vancomycin resistance gene. The study also demonstrates phenotypic as well as genotypic heterogeneity among isolates of VRE from clinical specimens.

Project description:BackgroundNosocomial infections caused by the bacterial pathogen Staphylococcus aureus can lead to serious complications due to the varying presence of secreted toxins. Comparative studies of genomic information and production rates are needed to assess the pathogenic potential of isolated strains. Genotypic and phenotypic profiling of clinical and colonising isolates of S. aureus was used to characterise the release of exotoxins. Blood isolates were compared with colonisation strains to determine similarities and differences of single strains and clusters.ResultsFifty-one fresh isolates obtained from colonised individuals (n = 29) and S. aureus bacteremia (SAB) patients (n = 22) were investigated. The prevalence of genes encoding for three cytolysins (alpha/beta/gamma toxin) and twenty-four superantigens (SEA-SElX) was determined. Isolates exhibited eighteen distinct combinations of superantigens. Sequence analysis identified mutated open reading frames in hla in 13.7% of all strains, in selw (92.2%) and in selx (15.7%). All corrupted genes were associated with specific clonal complexes. Functional assessment of alpha toxin activity by a rabbit erythrocyte lysis assay revealed that supernatants lacking alpha toxin still displayed hemolysis. This was due to the presence of gamma toxin, as proven by inhibition experiments using antisera raised against the respective recombinant proteins. Alpha toxin, SEC, and TSST1 production was quantified by enzyme-linked immunosorbent assays on supernatants of all hla, sec, and tst positive isolates. Blood isolates and colonising strains showed comparable amounts of secreted proteins within a wide range. Agr types I to IV were identified, but did not allow a prediction of high or low production rates. In contrast, alpha toxin production rates between distinct clonal complexes clearly differed. Spa typing was performed and revealed thirty-two unique spa gene patterns and eight small clusters comprising nineteen isolates. Recognised spa-typing clusters displayed highly similar production rates.ConclusionProduction rates of the three most prevalent exotoxins varied within both groups of blood isolates and colonising strains. By comparing genotypes and secretion, we found that identical complex gene patterns did not allow predictions of toxin production and function. However, identification of spa typing clusters was suitable to predict similar quantities of released exotoxins.

Project description:BackgroundHigh rates of dihydroartemisinin-piperaquine (DHA-PPQ) treatment failures have been documented for uncomplicated Plasmodium falciparum in Cambodia. The genetic markers plasmepsin 2 (pfpm2), exonuclease (pfexo) and chloroquine resistance transporter (pfcrt) genes are associated with PPQ resistance and are used for monitoring the prevalence of drug resistance and guiding malaria drug treatment policy.MethodsTo examine the relative contribution of each marker to PPQ resistance, in vitro culture and the PPQ survival assay were performed on seventeen P. falciparum isolates from northern Cambodia, and the presence of E415G-Exo and pfcrt mutations (T93S, H97Y, F145I, I218F, M343L, C350R, and G353V) as well as pfpm2 copy number polymorphisms were determined. Parasites were then cloned by limiting dilution and the cloned parasites were tested for drug susceptibility. Isobolographic analysis of several drug combinations for standard clones and newly cloned P. falciparum Cambodian isolates was also determined.ResultsThe characterization of culture-adapted isolates revealed that the presence of novel pfcrt mutations (T93S, H97Y, F145I, and I218F) with E415G-Exo mutation can confer PPQ-resistance, in the absence of pfpm2 amplification. In vitro testing of PPQ resistant parasites demonstrated a bimodal dose-response, the existence of a swollen digestive vacuole phenotype, and an increased susceptibility to quinine, chloroquine, mefloquine and lumefantrine. To further characterize drug sensitivity, parental parasites were cloned in which a clonal line, 14-B5, was identified as sensitive to artemisinin and piperaquine, but resistant to chloroquine. Assessment of the clone against a panel of drug combinations revealed antagonistic activity for six different drug combinations. However, mefloquine-proguanil and atovaquone-proguanil combinations revealed synergistic antimalarial activity.ConclusionsSurveillance for PPQ resistance in regions relying on DHA-PPQ as the first-line treatment is dependent on the monitoring of molecular markers of drug resistance. P. falciparum harbouring novel pfcrt mutations with E415G-exo mutations displayed PPQ resistant phenotype. The presence of pfpm2 amplification was not required to render parasites PPQ resistant suggesting that the increase in pfpm2 copy number alone is not the sole modulator of PPQ resistance. Genetic background of circulating field isolates appear to play a role in drug susceptibility and biological responses induced by drug combinations. The use of latest field isolates may be necessary for assessment of relevant drug combinations against P. falciparum strains and when down-selecting novel drug candidates.

Project description:Vibrio cincinnatiensis is a halophilic species which has been found in marine and estuarine environments worldwide. The species is considered a rare pathogen for which the significance for humans is unclear. In this study, nine veterinary isolates were investigated that were obtained from domestic animals in Germany. The isolates were mostly recovered from abortion material of pigs, cattle, and horse (amnion or fetuses). One isolate was from a goose. A human clinical strain from a case of enteritis in Germany described in the literature was also included in the study. Whole-genome sequencing (WGS) of all isolates and MALDI-TOF MS (matrix-assisted-laser-desorption/ionization time-of-flight mass spectrometry) were performed to verify the species assignment. All strains were investigated for phenotypic traits including antimicrobial resistance (AMR), biochemical properties, and two virulence-associated phenotypes (hemolytic activity and resistance to human serum). WGS data and MS spectra confirmed that all veterinary isolates are closely related to the type strain V. cincinnatiensis NCTC12012. An exception was the human isolate from Germany which is related to the other isolates but could belong to another species. The isolates were similar in most biochemical phenotypes. Only one strain showed a very weak hemolytic activity against sheep erythrocytes, and serum resistance was intermediate in two strains. AMR phenotypes were more variable between the isolates. Resistances were observed against ß-lactams ampicillin and cefoxitin and against tetracycline and the sulfonamide antibiotics trimethoprim and sulfamethoxazole. Some acquired AMR genes were identified by bioinformatics analyses. WGS and MALDI-TOF MS data reveal a close relationship of the veterinary isolates and the type strain V. cincinnatiensis NCTC12012, which is a clinical human isolate. As the veterinary isolates of this study were mostly recovered from abortion material (amnions and fetuses), a zoonotic potential of the veterinary isolates seems possible.

Project description:Staphylococcus aureus has recently overtaken Pseudomonas aeruginosa as the most commonly recognized bacterial pathogen that infects the respiratory tracts of individuals with the genetic disease cystic fibrosis (CF) in the United States. Most studies of S. aureus in CF patient lung infections have focused on a few isolates, often exclusively laboratory-adapted strains, and how they are killed by P. aeruginosa Less is known about the diversity of S. aureus CF patient lung isolates in terms of both their virulence and their interaction with P. aeruginosa To begin to address this gap, we recently sequenced 64 clinical S. aureus isolates and a reference isolate, JE2. Here, we analyzed the antibiotic resistance genotypes, sequence types, clonal complexes, spa types, agr types, and presence/absence of other known virulence factor genes of these isolates. We hypothesized that virulence phenotypes of S. aureus, namely, toxin production and the mucoid phenotype, would be lost in these isolates due to adaptation in the CF patient lung. In contrast to these expectations, we found that most isolates can lyse both rabbit and sheep blood (67.7%) and produce polysaccharide (69.2%), suggesting that these phenotypes were not lost during adaptation to the CF lung. We also identified three distinct phenotypic groups of S. aureus based on their survival in the presence of nonmucoid P. aeruginosa laboratory strain PAO1 and its mucoid derivative. Altogether, our work provides greater insight into the diversity of S. aureus isolates from CF patients, specifically the distribution of important virulence factors and their interaction with P. aeruginosa, all of which have implications in patient health.IMPORTANCEStaphylococcus aureus is now the most frequently detected recognized pathogen in the lungs of individuals who have cystic fibrosis (CF) in the United States, followed closely by Pseudomonas aeruginosa When these pathogens are found to coinfect the CF lung, patients have a significantly worse prognosis. While P. aeruginosa has been rigorously studied in the context of bacterial pathogenesis in CF, less is known about S. aureus Here, we present an in-depth study of 64 S. aureus clinical isolates from CF patients, for which we investigated genetic diversity utilizing whole-genome sequencing, virulence phenotypes, and interactions with P. aeruginosa We found that S. aureus isolated from CF lungs are phylogenetically diverse; most retain known virulence factors and vary in their interactions with P. aeruginosa (i.e., they range from being highly sensitive to P. aeruginosa to completely tolerant to it). Deepening our understanding of how S. aureus responds to its environment and other microbes in the CF lung will enable future development of effective treatments and preventative measures against these formidable infections.

Project description:Background and aimSubclinical mastitis (SCM) caused by erythromycin-resistant Staphylococcus aureus is a significant disease in lactating animals. Therefore, it is crucial to understand the genetic factors contributing to erythromycin resistance in S. aureus. This study aimed to estimate the prevalence of S. aureus in milk from subclinical mastitic cattle and buffaloes and tank milk samples as identified by probe-based real-time polymerase chain reaction (PCR) and the genotypic assessment of macrolide and erythromycin resistance profiles, as well as to analyze the phylogenetic relatedness of our local isolates of S. aureus.Materials and methodsIn total, 285 milk samples were analyzed using the California mastitis test to detect SCM. Milk samples were cultured on different specific Staphylococcus media. The presence of S. aureus was confirmed by Gram staining, the catalase and coagulase tests, the detection of hemolytic activity, DNase agar testing, and biofilm activity in Congo red medium. The genotypic identification of S. aureus (nuc) was performed. The determinants of erythromycin (ermA, ermB, ermC, and ermT) and macrolide resistance (msrA) were screened in all isolates. DNA sequencing of our local isolates of S. aureus was used to analyze their phylogenetic relatedness. Moreover, histopathological examination of tissue specimens of mammary gland was performed.ResultsThe S. aureus positivity rates were 36.4%, 48.8%, and 63.6% in cattle, buffalo, and bulk tank milk, respectively. Probe-based real-time PCR molecularly confirmed all 62 S. aureus isolates. Thirty-one isolates were subjected to PCR to create profiles of their genotypic erythromycin resistance. ermA, ermB, ermC, and ermT were present in 5 (8%), 26 (41.9%), 18 (29%), and 15 (24.1%) S. aureus isolates, respectively. Moreover, msrA was found in three (4.8%) strains. Eight PCR products were produced using standard PCR for DNA sequencing. Multiple sequence alignment, phylogenetic tree construction, and analysis of nuc in S. aureus revealed a high degree of homology (100%) with S. aureus strains isolated from milk in cases of bovine mastitis in India and Kenya. Histological analysis of udder tissues revealed extensive aggregation of mononuclear inflammatory cells in the interstitial connective tissue, primarily lymphocytes, and macrophages.ConclusionThis study showed a high prevalence of erythromycin resistance in S. aureus isolates. This information is vital for controlling mastitis and the spread of resistance genes between bacterial strains and hosts. Moreover, the probe-based real-time PCR approach is helpful for the rapid screening of S. aureus isolates and the consequent efficient treatment and control of S. aureus mastitis.

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Phenotypic and genotypic characterization of clinical Staphylococcus aureus isolates from Kenya.

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Phenotypic and genotypic characterization of clinical Staphylococcus aureus isolates from Kenya.

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