MLST Typing Research Articles

Transmission of MDR MRSA between primates, their environment and personnel at a United States primate centre.

MDR MRSA isolates cultured from primates, their facility and primate personnel from the Washington National Primate Research Center were characterized to determine whether they were epidemiologically related to each other and if they represented common local human-associated MRSA strains. Human and primate nasal and composite environmental samples were collected, enriched and selected on medium supplemented with oxacillin and polymyxin B. Isolates were biochemically verified as Staphylococcus aureus and screened for the mecA gene. Selected isolates were characterized using SCCmec typing, MLST and WGS. Nasal cultures were performed on 596 primates and 105 (17.6%) were MRSA positive. Two of 79 (2.5%) personnel and two of 56 (3.6%) composite primate environmental facility samples were MRSA positive. Three MRSA isolates from primates, one MRSA from personnel, two environmental MRSA and one primate MSSA were ST188 and were the same strain type by conventional typing methods. ST188 isolates were related to a 2007 ST188 human isolate from Hong Kong. Both MRSA isolates from out-of-state primates had a novel MLST type, ST3268, and an unrelated group. All isolates carried ≥1 other antibiotic resistance gene(s), including tet(38), the only tet gene identified. ST188 is very rare in North America and has almost exclusively been identified in people from Pan-Asia, while ST3268 is a newly reported MRSA type. The data suggest that the primate MDR MRSA was unlikely to come from primate centre employees. Captive primates are likely to be an unappreciated source of MRSA.

Confirmation of a Campylobacteriosis Outbreak Associated with Chicken Liver Pâté Using PFGE and WGS.

In May 2012, an outbreak of campylobacteriosis occurred in southern Sweden at a wedding reception affecting 44 persons. A total of 17 cases were notified (13 were culture positive for Campylobacter spp.). Epidemiological investigation suspected chicken liver pâté as the source of infection. The liver pâté had been deliberately undercooked, lightly fried to keep the right texture and mixed with spices. Campylobacter isolates from six cases as well as three Campylobacter isolates from chicken flocks previously raised by the producer delivering the liver were subtyped using pulsed-field gel electrophoresis and whole-genome sequencing. Indistinguishable PFGE profiles were identified among five human and one chicken C.jejuni isolates as well among the two C.coli isolates, one from a human case and one from a chicken. WGS supported the PFGE findings; the six C.jejuni isolates belonged to one cluster. All these six isolates were of MLST type ST 50 (ST-CC 21). This study highlights the importance of a combination of strict biosecurity at the flock-level as well as adequate cooking of chicken liver to prevent transmission of Campylobacter to humans.

Clonal diversity and epidemiological characteristics of Staphylococcus aureus: high prevalence of oxacillin-susceptible mecA-positive Staphylococcus aureus (OS-MRSA) associated with clinical isolates in Brazil.

BackgroundStaphylococcus aureus is the major cause of global and nosocomial infections with a significant impact in hospitals worldwide. Our objective was to investigate clinical and molecular characteristics of S. aureus isolates causing infections in patients admitted to hospitals from Recife city, Brazil, and investigate the prevalence of oxacillin-susceptible mecA-positive S. aureus (OS-MRSA) in the region, as well as genetically characterize the isolates and compare with epidemic clones.ResultsWe characterized 89 isolates in total, 31 clinical methicillin-resistant S. aureus (MRSA) and 58 methicillin-sensitive (MSSA) isolates by PFGE, MLST, spa typing and SCCmec genotyping. Isolates belonging to international MRSA clones were present: Brazilian epidemic clone (BEC) (61 % of MRSA isolates), Paediatric (36 %), New York/Japan (3 %). Some MSSA isolates were related to MRSA clones: USA400-related (10 % of MSSA isolates), Berlin clone (2 %), Paediatric (14 %), New York/Japan (2 %) and Southwest Pacific clone (17 %). MLST revealed new sequence types (ST’s): ST2381, ST2382, and ST2383 and new spa types: 10548 and 10550. Among isolates phenotypically identified as MSSA by antimicrobial susceptibility assays, we verified 30 oxacillin-susceptible isolates, which exhibited the mecA gene, without mec complex amplification and were thus classified as OS-MRSA. We observed clonal spread of MRSA and MSSA, including OS-MRSA, within several areas of the main hospital investigated and closely related isolates between hospitals analyzed.ConclusionsThe results of this study suggest a possible spread of the strains in hospital environment that could be responsible for nosocomial infections. We documented the presence of several MRSA clones, as well as new MLST and spa types, that were responsible for severe infections in hospitalized patients. The finding of OS-MRSA isolates could have implications for therapy, because testing for mecA and PBP2a is not a routine procedure performed by clinical microbiology laboratories in Brazil and, as consequence, these isolates could be misclassified as MSSA. Our data alert to the necessity to develop more effective strategies for epidemiological control of S. aureus in order to avoid an increase of hospital infections provoked by this pathogen. We reinforce the use of genetic methods, in addition to phenotypic tests, for a precise identification of MRSA.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0733-4) contains supplementary material, which is available to authorized users.

Epidemiological typing of Neisseria gonorrhoeae and detection of markers associated with antimicrobial resistance directly from urine samples using next generation sequencing

ObjectivesTo investigate the potential for next generation sequencing (NGS) to be used directly on clinical specimens that have tested positive for Neisseria gonorrhoeae by nucleic acid amplification testing (NAAT), to...

Trends and molecular mechanisms of antimicrobial resistance in clinical staphylococci isolated from companion animals over a 16 year period.

The objective of this study was to investigate the evolution of resistance to antimicrobials, corresponding mechanisms and molecular characteristics of Staphylococcus spp., between 1999 and 2014. Susceptibility to 38 antimicrobials was determined for 632 clinical staphylococcal isolates obtained from companion animals (dogs, cats, horses and other animals). Twenty antimicrobial resistance genes, including mecA and mecC, were screened by PCR. Methicillin-resistant staphylococci were characterized by spa (Staphylococcus aureus), SCCmec, MLST and PFGE typing. Statistical analyses were performed using SAS v9.3 and differences were considered relevant if P ≤ 0.05. The mecA gene was identified in 74 staphylococcal isolates (11.6%): 11 MRSA (40.7%), 40 methicillin-resistant Staphylococcus pseudintermedius (MRSP; 8.7%) and 23 methicillin-resistant CoNS (26.7%). Resistance to the majority of antimicrobials and the number of mecA-positive isolates increased significantly over time. Eighteen spa types were identified, including two new ones. MRSA isolates were divided into three PFGE clusters that included ST22-IV, ST105-II, ST398-V and ST5-VI. Most methicillin-resistant Staphylococcus epidermidis isolates were of clonal complex (CC) 5, including a new ST, and clustered in eight PFGE clusters. MRSP were grouped into five PFGE clusters and included ST45-NT, ST71-II-III, ST195-III, ST196-V, ST339-NT, ST342-IV and the new ST400-III. Methicillin-resistant Staphylococcus haemolyticus clustered in two PFGE clusters. The significant increase in antimicrobial-resistant and mecA-positive isolates in recent years is worrying. Furthermore, several isolates are MDR, which complicates antimicrobial treatment and increases the risk of transfer to humans or human isolates. Several clonal lineages of MRSA and methicillin-resistant S. epidermidis circulating in human hospitals and the community were found, suggesting that companion animals can become infected with and contribute to the dissemination of highly successful human clones. Urgent measures, such as determination of clinical breakpoints and guidelines for antimicrobial use, are needed.

Evolutionary dynamics of Enterococcus faecium reveals complex genomic relationships between isolates with independent emergence of vancomycin resistance.

Enterococcus faecium, a major cause of hospital-acquired infections, remains problematic because of its propensity to acquire resistance to vancomycin, which currently is considered first-line therapy. Here, we assess the evolution and resistance acquisition dynamics of E. faecium in a clinical context using a series of 132 bloodstream infection isolates from a single hospital. All isolates, of which 49 (37 %) were vancomycin-resistant, underwent whole-genome sequencing. E. faecium was found to be subject to high rates of recombination with little evidence of sequence importation from outside the local E. faecium population. Apart from disrupting phylogenetic reconstruction, recombination was frequent enough to invalidate MLST typing in the identification of clonal expansion and transmission events, suggesting that, where available, whole-genome sequencing should be used in tracing the epidemiology of E. faecium nosocomial infections and establishing routes of transmission. Several forms of the Tn1549-like element–vanB gene cluster, which was exclusively responsible for vancomycin resistance, appeared and spread within the hospital during the study period. Several transposon gains and losses and instances of in situ evolution were inferred and, although usually chromosomal, the resistance element was also observed on a plasmid background. There was qualitative evidence for clonal expansions of both vancomycin-resistant and vancomycin-susceptible E. faecium with evidence of hospital-specific subclonal expansion. Our data are consistent with continuing evolution of this established hospital pathogen and confirm hospital vancomycin-susceptible and vancomycin-resistant E. faecium patient transmission events, underlining the need for careful consideration before modifying current E. faecium infection control strategies.

<i>Yersinia pseudotuberculosis </i>Typing Using Multi-Locus Variable-Number Tandem Repeat Analysis

Estimated has been efficacy of MLVA25 method, used for differentiation and clusterization of Yersinia pestis strains, in reference to Yersinia pseudotuberculosis strains, as well as the search of Y. pseudotuberculosis strains mostly closely related to Y. pestis . Applied was the reduced version of the technique deploying 14 out of 25 VNTR loci. Comparative study of 71 Y. pseudotuberculosis isolates and five Y. pestis strains by means of MLVA25 and MLVA14 variants revealed 75 and 54 genotypes, respectively. Y. pseudotuberculosis strains of certain serovars corresponded to certain clusters of MLVA types. According to MLVA14 typing of 221 Y. pseudotuberculosis isolates the clusters including the strains belonging to MLST types ST19 (serovar O:3) and ST43 (serovar O:1b) were the most closely related to Y. pestis .

Methicillin-resistant Staphylococcus aureus (MRSA) in sheep and goat bulk tank milk from Southern Italy

Methicillin-resistant Staphylococcus aureus (MRSA), an important widespread cause of severe infection in both humans and animals, is a pathogen of significant public health concern. In this paper the prevalence of MRSA in sheep and goat bulk tank milk from southern Italy was assessed and nasal swabs from people working on the positive farms were collected. MRSA isolates from milk and nasal swabs were characterized by spa typing, MLST and SCCmec typing and tested for antimicrobial resistance. MRSA was detected in 2 of the 162 (1.23%) bulk tank samples analysed: the first from a sheep farm (Farm 1) and the second from a goat farm (Farm 2). As for farm personnel, no MRSA was detected in the unique nasal swab from Farm 1; while MRSA was detected in all the 3 nasal swabs from Farm 2. The MRSA isolate from Farm 1 was spa type t127, ST1, SCCmec IVa. The MRSA isolates from bulk tank milk and humans in Farm 2 showed an identical genetic profile: spa type t1255, ST398, SCCmec V. In Farm 2 all the isolates had the characteristics of general multidrug resistance and the milk and human MRSA had the same antimicrobial resistance pattern. Although the results showed low prevalence of MRSA in sheep and goat milk from southern Italy, the detection of recognized zoonotic genotypes in milk of both farms and the isolation in people working in one of the positive farm of MRSA strains with a genetic profile identical to that of MRSA from milk, emphasize the public health concern and highlight the need for additional surveillance including the detection of MRSA.

The Role of Glutamate Dehydrogenase (GDH) Testing Assay in the Diagnosis of Clostridium difficile Infections: A High Sensitive Screening Test and an Essential Step in the Proposed Laboratory Diagnosis Workflow for Developing Countries like China.

The incidence and severity of Clostridium difficile infection (CDI) in North America and Europe has increased significantly since the 2000s. However, CDI is not widely recognized in China and other developing countries due to limited laboratory diagnostic capacity and low awareness. Most published studies on laboratory workflows for CDI diagnosis are from developed countries, and thus may not be suitable for most developing countries. Therefore, an alternative strategy for developing countries is needed. In this study, we evaluated the performance of the Glutamate Dehydrogenase (GDH) test and its associated workflow on 416 fecal specimens from suspected CDI cases. The assay exhibited excellent sensitivity (100.0%) and specificity (92.8%), compared to culture based method, and thus could be a good screening marker for C. difficile but not for indication of toxin production. The VIDAS CDAB assay, which can detect toxin A/B directly from fecal specimens, showed good specificity (99.7%) and positive predictive value (97.2%), but low sensitivity (45.0%) and negative predictive value (88.3%), compared with PCR-based toxin gene detection. Therefore, we propose a practical and efficient GDH test based workflow strategy for the laboratory diagnosis of CDI in developing countries like China. By applying this new workflow, the CDI laboratory diagnosis rate was notably improved in our center, yet the increasing cost was kept at a minimum level. Furthermore, to gain some insights into the genetic population structure of C. difficile isolates from our hospital, we performed MLST and PCR toxin gene typing.

Molecular characterisation of acquired and overproduced chromosomal blaAmpC in Escherichia coli clinical isolates

Escherichia coli recovered from three hospitals in Barcelona (Spain) were studied to determine the prevalence of isolates with acquired AmpC (ac-AmpC) and/or overproduced chromosomal AmpC (c-AmpC). Mechanisms involved in blac-AmpC overexpression, blaac-AmpC and the plasmids associated with their distribution as well as the prevalence of plasmid-mediated quinolone resistance (PMQR) in AmpC-producing isolates were also determined. Isolates were selected according to their resistance phenotype. blaac-AmpC, alterations in the blac-AmpC promoter/attenuator, and PMQR genes [qnrA, qnrB, qnrS, aac(6′)-Ib-cr and qepA] were characterised by PCR and sequencing. blac-AmpC expression was determined by qRT-PCR. Population structure analysis was performed using PFGE, MLST and phylogenetic group PCR. Plasmids carrying blaac-AmpC were characterised by PCR-based replicon typing and S1-PFGE. IncI1 and IncF plasmids were also analysed by plasmid MLST and replicon sequence typing, respectively. Among 21563 E. coli isolates, 240 (1.1%) overproduced AmpC β-lactamases, including 180 (75.0%) harbouring ac-AmpC (132 CMY-2 variants and 48 DHA-1) and 60 (25.0%) c-AmpC enzymes. Three mutation profiles in the blac-AmpC promoter/attenuator were associated with a 72.5-, 19.9- and 5.8-fold increased expression, respectively. Moreover, 63.3% of ac-AmpC and 43.3% of c-AmpC isolates belonged to B2, D, E or F phylogenetic groups. PMQR was found in 31% of ac-AmpC isolates [38 qnrB4, 8 aac(6′)-Ib-cr, 6 qnrS1 and 3 qnrB19] and in 10% of c-AmpC isolates [5 aac(6′)-Ib-cr and 1 qnrS1]. IncI1-ST12 and IncF were associated with blaCMY-2 and blaDHA-1, respectively. These results suggest that ac-AmpC β-lactamases were the main mechanism of AmpC production. Isolates and plasmids both showed high genetic diversity.

Emergence of OXA-48-Producing Klebsiella pneumoniae in Taiwan.

The isolation of OXA-48-producing Enterobacteriaceae has increased dramatically in Mediterranean countries in the past 10 years, and has recently emerged in Asia. Between January 2012 and May 2014, a total of 760 carbapenem non-susceptible Klebsiella pneumoniae (CnSKP) isolates were collected during a Taiwan national surveillance. Carbapenemases were detected in 210 CnSKP isolates (27.6%), including 162 KPC-2 (n = 1), KPC-3, KPC-17, and NDM-1 (n = 1 each), OXA-48 (n = 4), IMP-8 (n = 18), and VIM-1 (n = 24). The four bla OXA-48 CnSKP isolates were detected in late 2013. Herein we report the emergence OXA-48-producing K. pneumoniae isolates in Taiwan. PFGE analysis revealed that the four isolates belonged to three different pulsotypes. Three isolates harboured bla CTX-M genes and belonged to MLST type ST11. In addition, the plasmids belonged to the incompatibility group, IncA/C. One isolate belonged to ST116 and the plasmid incompatibility group was non-typeable. The sequence upstream of the bla OXA-48 gene in all four isolates was identical to pKPOXA-48N1, a bla OXA-48-carrying plasmid. This is the first report of OXA-48-producing Enterobacteriaceae in Taiwan and the second report to identify bla OXA-48 on an IncA/C plasmid in K. pneumoniae. Given that three isolates belong to the same pandemic clone (ST11) and possess the IncA/C plasmid and similar plasmid digestion profile that indicated the role of clonal spread or plasmid for dissemination of bla OXA-48 gene, the emergence of OXA-48-producing K. pneumoniae in Taiwan is of great concern.

Bloodstream Infections due to Enterobacteriaceae Among Neonates in Poland – Molecular Analysis of the Isolates

Bloodstream infections (BSIs) are associated with a significantly increased risk of fatality. No report has been found about the molecular epidemiology of Enterobacteriaceae causing BSI in neonates in Poland. The aim of this work was to determine the antibiotic resistance profiles, virulence gene prevalence, the epidemiological and genetic relationships among the isolates from Enterobacteriaceae causing BSI in neonates with birth weight < 1501 g. Antimicrobial susceptibility testing was performed. PCR was performed to identify the presence of common beta-lactamase genes, virulence genes. PFGE and MLST were performed. The surveillance group contained 1,695 newborns. The incidence rate for BSIs was 5.9%, the fatality rate 15%. The most common species were Escherichia coli (n = 24) and Klebsiella pneumoniae (n = 16). CTX-M-15 was found in 6 E. coli, 8 K. pneumoniae, 1 Enterobacter cloacae strains. Among E. coli fimH (83.3%), ibeA (37.5%), neuC (20.8%) were the most frequent. PFGE demonstrated unique pulsotypes among E. coli. E. coli ST131 clone was found in 7 E. coli strains. PFGE of 16 K. pneumoniae strains showed 8 pulsotypes. Five isolates from one NICU belonged to one clone. MLST typing revealed 7 different ST with ST336 as the most prevalent. This study provides information about resistance, virulence and typing of Enterobacteriaceae strains causing BSI among neonates. E. coli and Klebsiella spp. isolated in this study have completely different epidemiology from each other.

Antibiotic resistance and mecA characterization of coagulase-negative staphylococci isolated from three hotels in London, UK.

Antibiotic resistance in bacteria isolated from non-healthcare environments, is a potential problem to public health. In our survey a total of 71 coagulase negative staphylococci (CNS) belonging to 11 different species were isolated from three large hotels in London, UK. The most prevalent species was Staphylococcus haemolyticus, with S. hominis, S. warneri, S. cohnii, and Staphylococcus epidermidis commonly detected. Antimicrobial susceptibilities and carriage of the mecA gene were determined for all of these isolates. Most (85.9%) staphylococci were resistant to multiple antibiotics with all displaying increased susceptibility toward penicillin, fusidic acid, erythromycin, and cefepime. Twenty-one (29.5%) of the isolates were mecA positive, however MIC values to oxacillin, normally associated with the carriage of mecA, varied widely in this group (from 0.06 to 256 mg/L). Fifteen of the twenty-one mecA positive isolates carried SCCmec of these seven were type V, one type I, one type II, and one type IV. Additionally, five of these 15 isolates carried a previously unreported type, 1A, which involves an association between class A mec complex and ccr type 1. The remaining six of the 21 isolates were non-typeable and carried a combination of class A mec complex and ccrC. In addition to this, we also report on new MLST types which were assigned for five S. epidermidis isolates. Four out of these five isolates had MICs between 0.06 and 256 mg/L to oxacillin and would be regarded as clinically susceptible but one isolate had a high oxacillin MIC of 256 mg/L. We demonstrated widespread multiple drug resistance among different staphylococcal species isolated from non-healthcare environments highlighting the potential for these species to act as a reservoir for methicillin and other forms of drug resistance.

Short tandem repeat genotyping for P. jirovecii is more sensitive to mixed genotype than MLST

Objectives The increasing reports of outbreaks in renal transplant and pediatric units highlight the need for reliable genotyping methods to characterize nosocomial acquisition of Pneumocystis jirovecii . PCR-based methods, mainly MLST, have been widely used in this setting. Recently, short tandem repeat (STR) typing was suggested to be more sensitive to mixed infection and detect minority genotypes. Till now, no comparison between MLST and STR have been performed. Methods Thirty-seven samples (one per patient) from two hospitals were analysed (Paris hospital, n = 18, Nantes hospital, n = 19). Each sample was analysed by both a STR typing assay using 6 nuclear markers as recently developed with a MLST typing scheme relying on 4 loci (2 nuclear: SOD and ITS1; and 2 mitochondrial genes: mtLSU , Cytb ) as recently proposed. Analyses were performed blindly for the other typing method. Results Thirty-two and 35 different genotypes were observed by MLST and STR typing, respectively. Mixtures of genotypes were detected in 6/37 (16.2%) samples and 20/37 (54.1%) in MLST and STR typing, respectively. One mixed infection out of 6 detected using MLST was not observed using STR typing. Overall, one STR marker tends to differentiate samples between Paris and Nantes hospitals (Chi 2 ; P = 0.003). We then analyzed the 18/37 (48.6%) samples with a unique P. jirovecii genotype. Out of the 18/37 (48.6%) samples with no mixture, 18 and 17 different genotypes were delineated using MLST and STR respectively (one STR genotype was recovered from 2 patients). Conclusion This study provides evidence that STR typing markers compared well to MLST for molecular typing of P. jirovecii from clinical samples. The present STRs is less time-consuming and better discriminated the geographical origin and detection of minority genotypes, which can impact on the interpretation of epidemiological data.

Molecular characterization of extended-spectrum beta-lactamases (ESBLs) produced by clinical isolates of Acinetobacter baumannii in Saudi Arabia.

BackgroundAcinetobacter baumannii is a common opportunistic pathogen that causes major nosocomial infections in hospitals. In this study, we hypothesized a high prevalence of A. baumanni ESBL (extended-spectrum beta-lactamase) among all collected isolates.MethodsA. baumannii isolates (n = 107) from ICU (Intensive care unit) of local hospitals in Makkah were phenotypically and genotypically characterized. The identity and antibiotic susceptibility of A. baumannii strains were determined using the Vitek-2 system. The identified ESBL producers were further analyzed by PCR and sequencing followed by MLST typing. blaTEM, blaSHV, and the blaCTX-M-group genes 1, 2, 8, 9, and 25 were investigated. Furthermore, blaOXA51-like and blaOXA23-like genes were also examined in the carbapenem-resistant A. baumannii isolates.ResultsOur data indicated a high prevalence of A. baumannii ESBL producers among the collected strains. Of the 107 A. baumannii isolates, 94 % were found to be resistant to cefepime and ceftazidime, and aztreonam using the Vitek 2 system. The genes detected encoded TEM, OXA-51-like and OXA-23-like enzymes, and CTX-M-group proteins 1, 2, 8, 9, and 25. MLST typing identified eight sequence type (ST) groups. The most dominant STs were ST195 and ST557 and all of them belong to worldwide clonal complex (CC) 2.ConclusionsThis study has shown that there is a high prevalence of antimicrobial resistance in A. baumannii. The diversity of STs may suggest that new ESBL strains are constantly emerging. The molecular diversity of the ESBL genes in A. baumannii may have contributed to the increased antimicrobial resistance among all isolates.

Analysis of the features of 45 identified CRISPR loci in 32 Staphylococcus aureus

Staphylococcus aureus (S. aureus) is a common pathogen that can cause serious infections, even death. Because of the horizontal gene transfer (HGT) of antibiotic resistance genes, the drug resistant condition is becoming increasingly prevalent. Recently, an adaptive immunity system, named clustered regularly interspaced short palindromic repeats (CRISPR), was discovered and demonstrated to confer a defense against foreign invading elements that may carry the antibiotic resistance genes. In this study, we reveal the features of 45 identified CRISPR loci and the CRISPR associated gene (Cas) in 32 S. aureus strains from CRISPR database. Five spacers of S. aureus 08BA02176 and MSHR1132 were homologous with foreign genetic sequences from phages or plasmids, even containing a spacer sequence identical to part of some phages' genomes containing lukPV gene that encodes the PVL toxin. Many S. aureus strains with the same CRISPR type shared the same MLST type. CRISPR loci that had 3 or more similar protein loci mostly belonged to the same CRISPR type. We came to the conclusion that the CRISPR/Cas of strains 08BA02176 and MSHR1132 were inherited from a common ancestor or recombined from Staphylococcus lugdunensis. CRISPR loci can be mobilized and can transfer among different but closely related species, and the same types of MLST strains exhibit a higher affinity to the same types of CRISPR loci. Bacteriophages may be the predominant challenge facing S. aureus. The CRISPR/Cas structure may limit the transmission of bacterial virulence among S. aureus.

Comparative proteomic analysis of extracellular proteins expressed by various clonal types of Staphylococcus aureus and during planktonic growth and biofilm development.

Staphylococcus aureus is well known for its biofilm formation with rapid emergence of new clones circulating worldwide. The main objectives of the study were (1) to identify possible differences in protein expression among various and closely related clonal types of S. aureus, (2) to establish the differences in protein expression in terms of size of protein spots and its intensities between bacteria which are grown statically (biofilm formation) with that of under aeration and agitation, and (3) to compare the differences in protein expression as a function of time (in hours). In this study, we selected six clinical isolates comprising two similar (MRSA-527 and MRSA-524) and four different (MRSA-139, MSSA-12E, MSSA-22d, and MSSA-10E) types identified by spa typing, MLST and SCCmec typing. We performed 2D gel migration comparison. Also, two MRSA isolates (527 and 139) were selected to determine quantitative changes in the level of extracellular proteins at different biofilm growth time points of 12, 24, and 48 h. The study was done using a strategy that combines 2-DGE and LC-MS/MS analysis for absolute quantification and identification of the extracellular proteins. The 2DGE revealed that the proteomic profiles for the isolates belonging to the similar spa, MLST, and SCCmec types were still quite different. Among the extracellular proteins secreted at different time points of biofilm formation, significant changes in protein expression were observed at 48 h incubation as compared to the exponential growth at 12 h incubation. The main conclusion of the work is that the authors do observe differences among isolates, and growth conditions do influence the protein content at different time points of biofilm formation.

WS19.2 Staphylococcus aureus population from cystic fibrosis patients in Spain: Results from a point-prevalence multicentre study

Objectives To characterize Staphylococcus aureus isolates colonising the lung of Spanish cystic fibrosis (CF) patients. Methods A prospective, multicentre study was performed (March-Nov. 2013) covering 341 CF-patients (180 adults, 161 children) from 17 hospitals. Respiratory samples (one per patient) and clinical and microbiological data were sent to our centre. S. aureus isolates were identified (MALDI-TOF MS) and antibiotic susceptibility (MicroScan) determined. Genetic relationships between the isolates were assessed by PFGE- Sma I, spa -typing and MLST, including a concatenate analysis of the alleles. SCC mec and agr type were characterized in methicillin-resistant S. aureus (MRSA). Results A total of 206 S. aureus isolates (60%) were obtained in 97 adults and 109 children. Prevalence in the different CF-Units varied between 13.3–86.6%. Pseudomonas aeruginosa co-colonisation was observed in 36 patients. Twenty-eight MRSA (13.7%) were detected in 11 Units, corresponding to SCCmec-IVc (22/28), SCCmec-IVa (3/28), and SCCmec-V (3/28). Eighty-seven isolates displaying unrelated PFGE were further analysed, showing a high genetic diversity by spa (the most frequent were CC002, CC012) and MLST (CC5, CC8, CC30, and CC45) typing. Three isolates grouped into the ST398, linked to MRSA carriage in pigs. Glycopeptide resistance was absent, whereas a cfr -mediated-linezolid resistant MRSA isolate was detected. Conclusion Overall prevalence of S. aureus in the Spanish CF-population was 60%, being 13.7% of the isolates MRSA. Genetic relationship between isolates was scarce although some widespread nosocomial and community clones were detected, including ST398 from animal origin.

Isolation, Virulence, and Antimicrobial Resistance of Methicillin-Resistant Staphylococcus aureus (MRSA) and Methicillin Sensitive Staphylococcus aureus (MSSA) Strains from Oklahoma Retail Poultry Meats.

Staphylococcus aureus is one the top five pathogens causing domestically acquired foodborne illness in the U.S. Only a few studies are available related to the prevalence of S. aureus and MRSA in the U.S. retail poultry industry. The objectives of this study were to determine the prevalence of S. aureus (MSSA and MRSA) in retail chicken and turkey meats sold in Tulsa, Oklahoma and to characterize the recovered strains for their antimicrobial resistance and possession of toxin genes. A total of 167 (114 chicken and 53 turkey) retail poultry samples were used in this study. The chicken samples included 61 organic samples while the rest of the poultry samples were conventional. The overall prevalence of S. aureus was 57/106 (53.8%) in the conventional poultry samples and 25/61 (41%) in the organic ones. Prevalence in the turkey samples (64.2%) was higher than in the chicken ones (42.1%). Prevalence of S. aureus did not vary much between conventional (43.4%) and organic chicken samples (41%). Two chicken samples 2/114 (1.8%) were positive for MRSA. PFGE identified the two MRSA isolates as belonging to PFGE type USA300 (from conventional chicken) and USA 500 (from organic chicken) which are community acquired CA-MRSA suggesting a human based source of contamination. MLST and spa typing also supported this conclusion. A total of 168 Staphylococcus aureus isolates (101 chicken isolates and 67 turkey isolates) were screened for their antimicrobial susceptibility against 16 antimicrobials and their possession of 18 different toxin genes. Multidrug resistance was higher in the turkey isolates compared to the chicken ones and the percentage of resistance to most of the antimicrobials tested was also higher among the turkey isolates. The hemolysin hla and hld genes, enterotoxins seg and sei, and leucocidins lukE-lukD were more prevalent in the chicken isolates. The PVL gene lukS-lukF was detected only in chicken isolates including the MRSA ones. In conclusion, S. aureus is highly prevalent in poultry retail meats sold in Oklahoma with a very low presence of human-originated MRSA. Multidrug resistance is not only prevalent in the MRSA isolates, but also in many MSSA poultry strains, particularly turkey.

Population Structure and Development of Resistance to Hymexazol Among Fusarium solani Populations from Date Palm, Citrus and Cucumber

AbstractA study was conducted to investigate genetic diversity and sensitivity to hymexazol among 80 isolates of Fusarium solani complex obtained from date palm (30), citrus (31) and cucumber (19). Characterization based on sequences of the EF1α and ITS rRNA showed that isolates belong to F. solani complex MLST type 3 + 4. AFLP analysis produced 980 polymorphic loci, 80 AFLP genotypes and moderate levels of genetic diversity (H = 0.2494). Clustering of the isolates was not related to the host or the geographical origin of the isolates. Analysis of molecular variance (amova) indicated the existence of a low level of genetic differentiation among populations obtained from different hosts (Fst = 0.0162) and regions (Fst = 0.0066). This may provide evidence for frequent movement of inoculum among hosts and regions in Oman, which could be attributed to cultural practices employed by farmers. Isolates of F. solani displayed variation in sensitivity to hymexazol, with EC50 values ranging from 2 to 5745 μg/ml (mean = 878 μg/ml); 19% of the isolates have an EC50 value of more than 1000 μg/ml. Findings are discussed in terms of the factors that affect diversity in F. solani isolates. The study reports for the first time the development of resistance to hymexazol among F. solani isolates from date palm, citrus and cucumber.