Molecular characterisation of methicillin-resistant Staphylococcus aureus strains

Methicillin-resistant Staphylococcus aureus (MRSA) is a pandemic human pathogen accounting for most of health-care associated infections throughout the world. However, in recent years, a more virulent strain of MRSA has emerged in the community defined as community-associated MRSA (CA-MRSA). These emerging strains of CA-MRSA are described to have different antibiotic susceptibility profiles, possess the SCCmec type IV element and usually produce the Panton-Valentine leukocidin (PVL) toxin. The majority of these CA-MRSA strains are associated with skin and soft tissue infections and necrotising pneumonia, with a 34% mortality rate. Identification and characterisation of MRSA isolates is mainly performed using phenotypic methods, which are time consuming. Little information exists on the prevalence and characteristics of MRSA isolates including antibiotic susceptibility patterns, PVL-producing CAMRSA strains, the SCCmec types and genotypes that might be circulating in the Steve Biko Academic Hospital. Identification and characterisation of MRSA isolates based on these criteria are important in controlling possible outbreaks in the clinical setting. In this study, 97 clinical MRSA isolates from the Steve Biko Academic Hospital, South Africa were collected between April 2006 to February 2007. These isolates were analysed and characterised using multiplex PCR (M-PCR), real-time PCR as well as staphylococcal protein A (spa) and hyper-variable region (HVR) typing. The aim of this study was to determine the antibiotic profiles, prevalence of MRSA isolates, the SCCmec types and the genotypes. Antibiotic susceptibility determination was performed using the disk diffusion susceptibility method as guidelined by the CLSI. Six distinct antibiotypes were identified with a total of 73%, 71%, 70% and 7% of MRSA isolates resistant to clindamycin, erythromycin, gentamicin and fusidic acid, respectively. The presence of Staphylococcus aureus specific 16S rRNA, the mecA and PVL genes was determined using a modified M-PCR assay. A total of 4% of the MRSA isolates possessed the PVL gene. Real-time PCR analysis also showed a 100% prevalence of the PVL gene in the same 4% MRSA isolates confirming the results of the first M-PCR assay. The second M-PCR was used to determine the SCCmec type prevalence and to distinguish between health-care associated MRSA (HA-MRSA) and CA-MRSA. SCCmec typing showed 67% of the isolates belonged to SCCmec type II and 14.4% SCCmec type III, both types belonging to HA-MRSA. A total of 4% of the MRSA isolates were CA-MRSA belonging to SCCmec type IVd. Genotyping results showed three distinct spa clusters whilst HVR showed six distinct clusters. Molecular-based assays proved to be useful tools to determine the prevalence and monitoring of MRSA outbreaks as well as to identify the SCCmec types, subtypes and genotypes of MRSA strains that might be circulating in the hospital. The determination of the different antibiotypes of MRSA can assist in the monitoring of the antibiotic resistant profile trends in the Steve Biko Academic Hospital, thus assisting with the correct implementation of antibiotic regimens for suspected MRSA infections. In an endeavour to assess the dissemination of MRSA strains especially PVL expressing CA-MRSA strains, it is of paramount importance to continuously monitor the emergence of these strains in clinical settings. CopyrightDissertation (MSc)--University of Pretoria, 2010.Medical Microbiologyunrestricte

Characterisation of Staphylococcus aureus bacteraemia at Tygerberg hospital

To elucidate the local epidemiology of Staphylococcus aureus bacteraemia, we characterised blood culture isolates using molecular methods and prospectively collected clinical data to determine the occurrence of community-acquired, methicillinresistant S. aureus (MRSA). Consecutive S. aureus blood culture isolates were collected over a one-year period from patients who were admitted to Tygerberg Academic Hospital in the Western Cape. A multiplex polymerase chain reaction (PCR) was used for the detection of spa, mecA and lukS/F-PV genes. Strain typing was performed using spa typing. Multiplex PCR for staphylococcal cassette chromosome mec (SCCmec) typing was also performed, as well as multilocus sequence typing (MLST) on selected isolates. Cases were categorised by clinical data as either hospital-acquired, healthcare-associated or community-acquired. One hundred and thirteen S. aureus isolates (30% MRSA) were collected from 104 cases of bacteraemia. According to clinical data, all community-acquired infections, 54% of hospital-acquired cases and the majority of healthcare-associated cases were due to methicillin-sensitive S. aureus (MSSA). Furthermore, all Panton-Valentine leukocidin (PVL)-positive isolates (15.9% of all S. aureus) were MSSA. MRSA strains were isolated from hospital-acquired cases (with a minority of healthcare-associated cases) and clustered mainly in spa-CC701 and CC012. SCCmec type IV was predominant. MLST clones included ST239-MRSAIII, ST36-MRSA-II and ST612-MRSA-IV. The predominant source for S. aureus bacteraemia was catheter-related infection (39%). Community-acquired S. aureus infections in our setting remain sensitive to methicillin and current treatment guidelines suffice. The majority of hospital-acquired and healthcare-associated infections were catheter-related. Prevention and treatment should be targeted accordingly. © SAJEI

A quantitative analysis of complexity of human pathogen-specific CD4 T cell responses in healthy M. tuberculosis infected South Africans

Author Summary: Human pathogen-specific immune responses are tremendously complex and the techniques to study them ever expanding. There is an urgent need for a quantitative analysis and better understanding of pathogen-specific immune responses. Mycobacterium tuberculosis (Mtb) is one of the leading causes of mortality due to an infectious agent worldwide. Here, we were able to quantify the Mtb-specific response in healthy individuals with Mtb infection from South Africa. The response is highly diverse and 66 epitopes are required to capture 80% of the total reactivity. Our study also show that the majority of the identified epitopes are restricted by multiple HLA alleles. Thus, technical advances are required to capture and characterize the complete pathogen-specific response. This study demonstrates further that the approach combining identified epitopes into "megapools" allows capturing a large fraction of the total reactivity. This suggests that this technique is generally applicable to the characterization of immunity to other complex pathogens. Together, our data provide for the first time a quantitative analysis of the complex pathogen-specific T cell response and provide a new understanding of human infections in a natural infection setting

Die molekulare charakterisierung des zwei komponenten-systems sae in staphylokokken und seiner rolle in der Regulation des Eap adhäsins unter SDS vermittelten stress bedingungen

The Staphylococcus aureus two component system (TCS) sae governs expression of numerous virulence factors, including Eap (extracellular adherence protein), which in turn among other functions also mediates invasion of host cells. The sae TCS is encoded by the saePQRS operon, with saeS coding for the sensor histidine kinase (SaeS) and saeR encoding the response regulator (SaeR). The saeRS system is preceded by two additional open reading frames (ORFs), saeP and saeQ, which are predicted to encode a lipoprotein (SaeP) and a membrane protein (SaeQ), respectively. Earlier, we have shown that SDS-containing subinhibitory concentrations of biocides (Perform®) and SDS alone activate sae transcription and increase cellular invasiveness in S. aureus strain Newman. The effect is associated with an amino acid exchange in the N-terminus of SaeS (L18P), specific to strain Newman. In this work, the role of whether the two additional genes, saePQ coding for the accessory proteins SaeP and SaeQ, respectively, are involved in SDS-mediated saeRS was investigated. It could demonstrated that the lack of the SaeP protein resulted in an increased saeRS transcription without SDS stress in both SaeSL/P variants, while the SDS effect was less pronounced on sae and eap expression compared to the Newman wildtype, suggesting that the SaeP protein represses the sae system. Also, SDS-mediated inductions of sae and eap transcription along with enhanced invasion were found to be dependent on presence of the SaeSP variant in Newman wildtype. On the other hand, the study also shows that the saePQ region of the sae operon is required for fully functional two-component system saeRS under normal growth conditions, but it is not involved in SDS-mediated activation of the saeS signaling and sae-target class I gene, eap. In the second approach, the study investigates whether SDS-induced sae expression and host cell invasion is common among S. aureus strains not carrying the (L18P) point mutation. To demonstrate this strain Newman, its isogenic saeS mutants, and various S. aureus isolates were analysed for sae, eap expression and cellular invasiveness. Among the strains tested, SDS exposure resulted only in an increase of sae transcription, Eap production and cellular invasiveness in strain Newman wild type and MRSA strain ST239-635/93R, the latter without an increase in Eap. Interestingly, the epidemic community-associated MRSA strain, USA300 LAC showed a biphasic response in sae transcription at different growth stages, which, however, was not accompanied by increased invasiveness. All other clinical isolates investigated displayed a decrease of the parameters tested. While in strain Newman the SDS effect was due to the saeSP allele, this was not the case in strain ST239-635/93R and the biphasic USA300 strains. Also, increased invasiveness of ST239-635/93R was found to be independent of Eap production. Furthermore, to investigate the global effect of SDS on sae target gene expression, strain Newman wild-type and Newman ∆sae were treated with SDS and analyzed for their transcription profiles of sae target genes using microarray assays. We could show that subinhibitory concentrations of SDS upregulate and downregulate gene expression of several signaling pathways involved in biosynthetic, metabolic pathways as well as virulence, host cell adherence, stress reponse and many hypothetical proteins. In summary, the study sheds light on the role of the upstream region saePQ in SDS-mediated saeRS and eap expression during S. aureus SDS stress. Most importantly, the study also shows that subinhibitory SDS concentrations have pronounced strain-dependent effects on sae transcription and subsequent host cell invasion in S. aureus, with the latter likely to be mediated in some strains by other factors than the known invasin Eap and FnBP proteins. Moreover, there seems to exist more than the saeSP-mediated mechanism for SDS-induced sae transcription in clinical S. aureus isolates. These results help to further understand and clarify virulence and pathogenesis mechanisms and their regulation in S. aureus.Das Zwei Komponenten-Systems (TCS) Sae in S. aureus reguliert die Expression einer Vielzahl von Virulenzfaktoren, dazu gehört unter anderem das extrazelluläre Adhärenzprotein Eap, welches neben weiteren Funktionen, die Invasion in eukaryotische Wirtszellen vermittelt. Die Gene des sae TCS sind in einem Operon organisiert (saePQRS), wobei saeS für die sensorische Histidinkinase (SaeS) und saeR für den „Response Regulator“ (SaeR) kodieren. Diesen Genen sind zwei weitere Genabschnitte, saeP und saeQ, vorangestellt, wobei saeP vermutlich für ein Lipoprotein (SaeP) und saeQ für ein Membranprotein (RelQ) kodieren. In einer früheren Arbeit konnten wir zeigen, dass SDS-haltige Biozide (Perform©) unter sub- inhibitorischen Konzentrationen, sowie reines SDS, die sae Transkription aktiviert und dadurch zu einer erhöhten Invasion des S. aureus Stamms Newman in Wirtszellen führt. Dieser Effekt ist assoziiert mit einem spezifischen Aminosäureaustausch im N-terminus von SaeS (L18P) des Stamm Newman. In dieser Arbeit soll nun die Beteiligung der zwei zusätzlichen Gene, saeP und saeQ, an der SDS vermittelten transkriptionellen Induktion von saeR/S untersucht werden. Es konnte gezeigt werden, dass ohne SaeP, die saeR/S Transkription in beiden SaeL/P Varianten erhöht war, wobei eine zusätzliche SDS Behandlung hierfür nicht notwendig war. Im Gegenteil, es zeigte sich, dass der SDS Effekt auf die sae und eap Expression in der saeP Mutante deutlich weniger ausgeprägt ist als im Wildtyp Stamm. Das läßt vermuten, dass das Lipoprotein SaeP repremierend auf das sae System einwirkt. Des Weiteren wurde festgestellt, dass die SDS vermittelte transkriptionelle Induktion von sae und eap, zusammen mit der erhöhten Invasion, abhängig vom vorhanden sein der SaeSP Variante im Newman Wildtyp Stamm ist. Die Arbeit zeigt, dass die saePQ Region wichtig ist für die vollständige Funktion des Zwei Komponenten Systems SaeRS unter normalen Wachstumsbedingungen. Jedoch ist diese Region nicht involviert in der Aktivierung von SaeS, mit SDS als Signalgeber, sowie der darauffolgenden Aktivierung des sae Zielgens eap. In einem zweiten Ansatz wurde untersucht, ob die SDS induzierte sae Expression und Wirtszellinvasion auch häufig in S. aureus Stämmen auftritt, welche keine (L18P) Punktmutation besitzen. Dafür wurde Stamm Newman, die isogene saeS Mutante und verschiedene S. aureus Klinikisolate auf ihre sae, eap Expression, sowie zelluläre Invasionsfähigkeit hin analysiert. Von den getesteten Stämmen reagiert nur Wildtyp Stamm Newman und ein MRSA Stamm ST239-635/93R mit gesteigerter sae Transkription, Eap Produktion und zellulärer Invasion. Der MRSA Stamm jedoch ohne erhöhte Eap Produktion. Interessanterweise zeigt der „community- associated“ MRSA Stamm USA300 LAC eine biphasische sae Transkription in verschiedenen Wachstumsphasen, welche jedoch nicht einhergeht mit erhöhter Invasion. Alle anderen Klinikisolate zeigten abnehmende Tendenzen in den getesteten Parametern. Während im Stamm Newman der SDS Effekt auf das saeSP Allel zurückzuführen ist, gilt dies nicht für den Stamm ST239-635/93R, sowie den biphasischen Stamm USA300. Außerdem konnte gezeigt werden, dass die erhöhte Invasion des Stamms ST239-635/93R unabhängig von seiner Eap Produktion ist. Des Weiteren zeigten wir den globalen Effekt von SDS auf die sae Zielgenexpression. Dafür behandelten wir Wildtyp Stamm Newman mit SDS und analysierten die Transkription der sae Zielgene mittels Microarray Analyse. Wir konnten zeigen, dass subinhibitorische SDS Konzentrationen, induzierende als auch repremierende Auswirkungen auf die Genexpression haben. Dabei sind Gene betroffen, die involviert sind in verschiedene Signalwege, Biosynthese/Metabolismus als auch in Virulenz, Wirtzelladhärenz und Stressantwort. Zusammenfassend gibt die Arbeit Aufschluss über die Rolle der „upstream“ Region saePQ hinsichtlich der SDS-abhängigen saeRS und eap Expression in S. aureus. Am wichtigsten ist hierbei die Erkenntnis, das subinhibitorische SDS Konzentrationen einen deutlichen stammabhängigen Effekt auf die sae Transkription und daraus folgernd auf die Wirtszellinvasion von S. aureus haben. Letzteres wird vermutlich in manchen Stämmen durch andere Faktoren als die bekannten Invasinproteine Eap und FnBP vermittelt. Außerdem scheint es in den klinischen S. aureus Isolaten mehr als nur den saeSP abhängigen Mechanismus der sae Induktion durch SDS zu geben. Diese Ergebnisse helfen uns die Virulenz und pathogenen Mechanismen als auch deren Regulation in S. aureus zu verstehen. Die Beobachtungen tragen zu unserem Verständnis bei, wie das sae System Signale der Umgebung detektieren kann. Dies ist bis jetzt eine Fragestellung mit vielen Unbekannten

Characterisation of <I>Staphylococcus aureus </I>bacteraemia at Tygerberg Hospital

Please help populate SUNScholar with the full text of SU research output. Also - should you need this item urgently, please send us the details and we will try to get hold of the full text as quick possible. E-mail to [email protected]. Thank you.Journal Articles (subsidised)Geneeskunde en GesondheidswetenskappeGeneeskundige Mikrobiologi

Prevalence of blaSHV, blaTEM and blaCTX-M antibiotic resistance genes in selected bacterial pathogens from the Pretoria Academic Hospital

Extended spectrum beta-lactamases (ESBLs) are considered one of the most important antibiotic resistance mechanisms. Multidrug resistance is emerging in many Gram-negative pathogens and is associated with severe nosocomial infections. The emergence of ESBL-producing bacteria coincided, in the 1980s, with the increased usage of cephalosporins. This study investigated the prevalence of ESBLs in 56 selected clinical bacterial isolates, collected over a three week period during August 2006, from the Pretoria Academic Hospital. Isolates included: one Citrobacter freundii; 13 Escherichia coli; three Morganella morganii ssp morganii; four Enterobacter cloacae; 34 Klebsiella pneumoniae and one Proteus penneri. Multiplex PCR was used to detect the presence of the blaSHV, blaTEM and blaCTX-M genes. The results were as follow for each of the isolates: i) E. coli: blaSHV detected in 8% (1/13); blaTEM and blaSHV detected in 15% (2/13); blaTEM and blaCTX-M detected in 23% (3/13) and blaTEM detected in 54% (7/13) of the isolates. ii) M. morganii: blaSHV detected in 33% (1/3) and blaTEM and blaCTX-M detected in 33% (1/3) of the isolates. The third M. morganii isolate was negative for all the genes. iii) The P. penneri isolate was positive for the blaTEM gene only. iv) K. pneumoniae: blaSHV detected in 3% (1/34); blaSHV and blaTEM detected in 6% (2/34); blaTEM detected in 15% (5/34) and blaTEM and blaCTX-M detected in 35% (12/34) of the isolates. v) E. cloacae: blaTEM detected in 25% (1/4); blaSHV and blaTEM detected in 25% (1/4); blaSHV and blaCTX-M detected in 25% (1/4) while the last isolate was negative for all three genes. The overall prevalence of these ESBL genes in this study was 48% (27/56). According to the literature these results were higher when compared to 33% for E. coli and 15% for K. pneumoniae in Europe and only 0.8% in Denmark for similar pathogens. These research findings indicated that it is crucial to routinely monitor the prevalence of these resistance genes in a hospital setting to ensure that antibiotic treatment regimens can be adjusted accordingly.Poster presented at the University of Pretoria Health Sciences Faculty Day, August 2008, Pretoria, South Afric

Prevalence of antibiotic resistance genes in Acinetobacter baumannii isolated from clinical specimens from Pretoria Academic Hospital

Acinetobacter baumannii is an important cause of nosocomial infections in hospitals, with most cases occurring in immunocompromised patients. Nosocomial infections are most often associated with medical device related infections due to biofilm formation. These bacteria are difficult to control due to the high resistance in these environments specifically due to their biofilm forming ability. A rise in the number of infections caused by A. baumannii in recent years together with the emergence of pan-drug resistant strains has been noted. An increase in carbapenem resistance worldwide limits the range of therapeutic alternatives in treating Acinetobacter-associated infections. The two main gene families responsible for this resistance are the Metallo-beta-lactamases (MBL’s) and the Carbapenem-hydrolysing-class-D-beta-lactamases (CHDL’s). The MBL genes confer resistance to all beta-lactams, except aztreonam. The CHDL’s are widespread beta-lactamases in A. baumannii and code for carbapenemase activity. These genes can either be plasmid or chromosomally encoded. This study screened for the following MBL genes: IMP, VIM, SIM, SPM and GIM, as well as the following CHDL genes: OXA-23, OXA-24, OXA-51 and OXA-58. The prevalence of these nine antibiotic resistance genes were determined in 97 clinical isolates of A. baumannii using two multiplex polymerase chain reactions (PCR’s). According to the results obtained, OXA-51 had a prevalence of 82% (80/97) and OXA-23 at 59% (57/97). There was only one isolate that was positive for a MBL gene (SPM). A study conducted in Turkey showed prevalence of 78% for OXA-51, which is lower than the 82% prevalence obtained in this study. However, prevalence of 91% was obtained in a study in Singapore for OXA-23, which is higher than the 59% prevalence obtained in this study. A similar study conducted in Brazil on Pseudomanas aeruginosa showed a prevalence of 20-45% for the SPM gene, which is higher than the 1% obtained in this study. According to the results obtained in this study, it is evident that there is an urgent need for further surveillance and monitoring of Acinetobacter baumannii due to the high prevalence of antibiotic resistance genes in this clinical setting.Poster presented at the University of Pretoria Health Sciences Faculty Day, 20 August 2008, Pretoria, South Africa

SDS interferes with SaeS signalling of Staphylococcus aureus independently of SaePQ

CITATION: Makgotlho, P. E. et al. 2013. SDS interferes with SaeS signalling of Staphylococcus aureus independently of SaePQ. PLoS ONE, 8(8): e71644, doi:10.1371/journal.pone.0071644.The original publication is available at http://journals.plos.org/plosoneThe Staphylococcus aureus regulatory saePQRS system controls the expression of numerous virulence factors, including extracellular adherence protein (Eap), which amongst others facilitates invasion of host cells. The saePQRS operon codes for 4 proteins: the histidine kinase SaeS, the response regulator SaeR, the lipoprotein SaeP and the transmembrane protein SaeQ. S. aureus strain Newman has a single amino acid substitution in the transmembrane domain of SaeS (L18P) which results in constitutive kinase activity. SDS was shown to be one of the signals interfering with SaeS activity leading to inhibition of the sae target gene eap in strains with SaeSL but causing activation in strains containing SaeSP. Here, we analyzed the possible involvement of the SaeP protein and saePQ region in SDS-mediated sae/eap expression. We found that SaePQ is not needed for SDS-mediated SaeS signaling. Furthermore, we could show that SaeS activity is closely linked to the expression of Eap and the capacity to invade host cells in a number of clinical isolates. This suggests that SaeS activity might be directly modulated by structurally non-complex environmental signals, as SDS, which possibly altering its kinase/phosphatase activity.http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0071644Publisher's versio

Prevalence of extended-spectrum-ß-lactamase (ESBL) and metallo-ß-lactamase (MBL) antibiotic resistance genes in Enterobacter species in Pretoria Academic Hospital

The prevalence of blaCTX-M, blaSHV and blaTEM extended-spectrum beta-lactamases (ESBLs) and VIM metallo-beta-lactamases (MBLs) producing Enterobacter are increasing worldwide. Multidrug resistance due to the presence of these resistance genes in the Enterobacteriaceae family is an important reason for therapy failure during treatment with 3rd generation cephalosporins and carbapenems. Methods for the detection of ESBL and MBL can be phenotypic or genotypic. Molecular techniques such as multiplex polymerase chain reaction (PCR) assays can be used for the simultaneous detection of various resistant genes. This study investigated the prevalence of ESBLs and MBLs antibiotic resistance genes in Enterobacter species in Pretoria Academic Hospital using a multiplex PCR assay, which simultaneously detected the blaCTX-M, blaSHV, blaTEM and VIM genes. Ninety seven (97) consecutive clinical Enterobacter isolates (16 E. aerogenes and 81 E. cloacae isolates) were collected from the diagnostic division of the Department of Medical Microbiology. The following results were obtained in E. aerogenes: blaCTX-M, blaTEM, blaSHV were detected in 13% (2/16) while blaCTX-M, blaTEM were detected in 13% (2/16) of the isolates. A total of 75% (12/16) of the isolates were negative for all three genes. In the E. cloacae isolates: blaCTX-M, blaTEM, blaSHV were detected in 7% (6/81) while blaCTX-M, blaTEM were detected in 28% (23/81) and blaTEM, blaSHV were detected in 20% (16/81). Only the blaTEM gene was detected in 6% (5/81) of the E. cloacae isolates. In 30% (21/81) of isolates none of the genes were identified. None of the Enterobacter isolates analysed in this study were positive for the VIM gene. According to the results obtained in this study, the prevalence of ESBL antibiotic resistance genes in Enterobacter species is 56% (54/97) in this clinical setting. It is therefore essential to include molecular technique as part of the surveillance to monitor the circulation of these resistant genes in a clinical setting.Poster presented at the University of Pretoria Health Sciences Faculty Day, August 2008, Pretoria, South Africa, as well as at the Department of Medical Microbiology, University of Pretoria, Pretoria, South Afric