What Is the Origin of Livestock-Associated Methicillin-Resistant Staphylococcus aureus Clonal Complex 398 Isolates from Humans without Livestock Contact? An Epidemiological and Genetic Analysis

Fifteen percent of all methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 398 (CC398) human carriers detected in The Netherlands had not been in direct contact with pigs or veal calves. To ensure low MRSA prevalence, it is important to investigate the likely origin of this MRSA of unknown origin (MUO). Recently, it was shown that CC398 strains originating from humans and animals differ in the presence of specific mobile genetic elements (MGEs). We hypothesized that determining these specific MGEs in MUO isolates and comparing them with a set of CC398 isolates of various known origin might provide clues to their origin. MUO CC398 isolates were compared to MRSA CC398 isolates obtained from humans with known risk factors, a MRSA CC398 outbreak isolate, livestock associated (LA) MRSA CC398 isolates from pigs, horses, chickens, and veal calves, and five methicillin-susceptible Staphylococcus aureus (MSSA) CC398 isolates of known human origin. All strains were spa typed, and the presence or absence of, scn, chp, φ3 int, φ6 int, φ7 int, rep7, rep27, and cadDX was determined by PCRs. The MRSA CC398 in humans, MUO, or MRSA of known origin (MKO) resembled MRSA CC398 as found in pigs and not MSSA CC398 as found in humans. The distinct human MSSA CC398 spa type, t571, was not present among our MRSA CC398 strains; MRSA CC398 was tetracycline resistant and carried no φ3 bacteriophage with scn and chp. We showed by simple PCR means that human MUO CC398 carriers carried MRSA from livestock origin, suggestive of indirect transmission. Although the exact transmission route remains unknown, direct human-to-human transmission remains a possibility as well.     

Distinction of Staphylococcal Cassette Chromosome mec type V elements from Staphylococcus aureus ST398

Methicillin resistant S. aureus (MRSA) is a major threat for human health and well-being. In recent years, it has become clear that livestock is a potential reservoir for MRSA, most livestock-associated isolates belonging to the ST398 lineage. Importantly, ST398 strains were also reported as causative agents of severe invasive infections in humans with no evidence for livestock associations. Here we document the sequence of the J1 region of the type V (5C2&5) SCCmec element and its right chromosomal junction in the clinical PVL-positive ST398 MRSA isolate UMCG-M4. Sequence comparisons show that this SCCmec element and related type V elements from other S. aureus isolates share a common core structure, but differ substantially in the so-called J1 region. Additional PCR analyses and typing studies indicate that the J1 region of strain UMCG-M4 is specific for SCCmec elements of PVL-positive ST398 isolates. Lastly, we show that the sequenced right chromosomal junction is invariant in strains of the ST398 lineage.     

Transmissibility of livestock-associated methicillin-resistant Staphylococcus aureus (ST398) in Dutch hospitals

We quantified nosocomial transmission rates of sequence type (ST) 398 methicillin-resistant Staphylococcus aureus (MRSA) (an emerging livestock-associated MRSA clone) and non-ST398 MRSA isolates in patients hospitalized without infection control measures in 51 Dutch hospitals. Identification of 174 index patients initiated 139 post-exposure screenings of 9925 persons. There were 65 genotype-confirmed secondary cases (three and 62 for ST398 and non-ST398 MRSA, respectively), yielding a relative transmission risk for ST398 MRSA of 0.28 (95% CI 0.09–0.90), which was not sensitive to adjustment for duration of hospitalization at time of detection. Nosocomial transmission of ST398 MRSA is 72% less likely than that of non-ST398 MRSA strains.     

Characterization of tetracycline and methicillin resistant Staphylococcus aureus strains in a Spanish hospital: Is livestock-contact a risk factor in infections caused by MRSA CC398?

Tetracycline-resistance (Tet^R) has been postulated as a marker of the livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) lineage CC398. Objectives of the study: to determine the spa-types and assigned MLST clonal complexes (CCs) among all 98 MRSA-Tet^R strains recovered during 2011–2012 (from different patients) in a Spanish Hospital, analyzing the possible correlation with livestock-contact of the patients. All 98 strains were assigned to 9 CCs: CC398 (60.2%), CC1 (19.4%), CC5 (12.2%), and other CCs (8.2%). The 98 patients were classified into three groups: (A) contact with livestock-animals (n = 25); (B) no-contact with livestock-animals (n = 42); (C) no information about animal contact (n = 31). A significant higher percentage of CC398 strains was obtained in group A (76%) than in group B (50%) (p < 0.05), being the percentage in group C of 61.3%. Most of MRSA-Tet^R-CC398 strains presented a multi-resistance phenotype, including erythromycin, clindamycin, and ciprofloxacin, and the most prevalent detected genes were tet(M) and erm(C). Three strains presented the phenotype macrolide-susceptibility/lincosamide-resistance and contained the vga(A) gene. MRSA-CC1 strains showed higher percentages of erythromycin/clindamycin resistance (95%/89%) than MRSA-CC398 strains (58%/63%), and this resistance was usually mediated by erm(C) gene. Most of MRSA-CC5 strains showed resistance to ciprofloxacin, tobramycin/kanamycin and erythromycin. None of the strains presented the genes lukF/lukS-PV, tsst-1, eta, etb or etd. All MRSA-CC398 strains lacked the genes of the immune-evasion-cluster, but MRSA-CC1 strains carried these genes (type E). In conclusion, although MRSA CC398 is detected in a significant higher proportion in patients with livestock-contact; its detection in people without this type of contact also indicates its capacity for human-to-human transmission.     

Prevalence of fibronectin-binding protein (FnbA and FnbB) genes among clinical isolates of methicillin resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

One of the most important stages in the occurrence of infection caused by Staphylococcus aureus is able to adhere of the bacterium to the cells and the extracellular matrix. Among adhesins, two fibronectinbinding proteins, (FnbA and FnbB) have been proved significantly to contribute to tissue colonization in various pathological conditions and indwelling medical device related infections. The aims of this study were to detect fnbA and fnbB genes and relation to the fibronectin binding ability in clinical isolates of methicillin resistant Staphylococcus aureus (MRSA). Of the 62 MRSA clinical isolates collected from selected hospitals in Tehran, Iran, fibronectin binding ability was determined by microtiter tissue culture coated by fibronectin plates. All MRSA isolates were examined for determination the fnbA and fnbB genes by using PCR method. The prevalence of fnbA and fnbB in MRSA strains was 82.2 and 46.7% respectively. In 26 (42%) MRSA strains both fnbA and fnbB genes were positive. All fnbA ⁺ or fnbB ⁺ strains were found to be positive also to in vitro fibronectin binding activity, while the fnb-negative strains were found negative to the in vitro binding assay. The finding that, fibronectin-adhesins are present in the most of the MRSA clinical isolates encourages the development of strategies to specifically block the interaction of this bacterium with fibronectin by antagonist molecules.     

Subdivision of MRSA CC398 isolates using MALDI-TOF MS

Outbreak investigations demand a fast and discriminative typing method. MALDI-TOF MS has been shown to be a rapid, easy and inexpensive method of subtyping MRSA.The aim of the present study is to explore whether it is possible to subdivide isolates of MRSA CC398, commonly livestock associated, using an enhanced version of the MALDI-TOF MS typing method that we previously described (Østergaard et al, 2015). We included MALDI-TOF spectra from 378 isolates of MRSA belonging to CC398, of which 322 were epidemiologically independent. We identified 17 peaks as discriminatorily useful and could therefore reliably subdivide the isolates into 23 subtypes, including a distinct type corresponding to a strain from an unusual and initially undiscovered hospital outbreak. Whole genome sequencing was carried out for 193 of the isolates and compared with both the spa type and an antibiogram of these strains. The proposed MALDI-TOF subdivision method for MRSA CC398 was found to be more discriminative than both spa typing and resistotyping, and had a high negative predictive value for ruling out a close genetic relationship between pairs of strains with different MALDI-TOF types. We conclude that the MALDI-TOF-based typing method can be used for rapid and inexpensive routine subdivision of MRSA belonging to CC398.     

Livestock-associated Staphylococcus aureus

Staphylococcus aureus has long been associated with livestock. Livestock can be carriers of S. aureus, but can also become infected. The best-known infection is bovine mastitis. The discovery of methicillin-resistant S. aureus belonging to sequence type (ST)398 boosted interest in livestock-associated S. aureus. ST398 is pandemic. Whole genome sequencing and other genetic analyses have shown that livestock-associated strains are distinct from human-derived strains. However, there is also an exchange of strains between the reservoirs. Livestock-associated and human-associated strains share virulence factors, but have also distinct virulence factors that appear to be important in host adaptation. Exchange of genes encoding these virulence factors between strains may expand the host range and thereby threaten public health. Vaccination of animals may be a solution to this problem, but new avenues for vaccination need to be explored, because no vaccine is currently available.     

Isolation and characterization of phages with lytic activity against methicillin-resistant Staphylococcus aureus strains belonging to clonal complex 398

Some years ago, MRSA clonal complex (CC) 398 emerged, which spread extensively in livestock animals. People in contact with food production animals are at high risk of colonization. A reduction of MRSA CC398 in livestock might be achieved by application of virulent phages. However, there have not yet been any reports published on phages lysing MRSA CC398 strains. In this study, three virulent phages (PSa1, PSa2 and PSa3) with lytic activity against MRSA CC398 strains were isolated from German pig farms. Morphologically, the phages are members of the family Podoviridae, and they exhibited an identical host range. They lysed 52 (60 %) out of 86 tested MRSA CC398 strains representing 18 different spa types. While the PSa1 and PSa3 genomes have a similar size of approximately 17.5 kb, the PSa2 genome is somewhat larger (ca. 18.5 kb). Southern hybridization revealed strong DNA homologies between the phages, which was confirmed by sequence analysis of cloned restriction fragments and PCR products. Moreover, the whole PSa3 genomic sequence (17,602 bp) showed a close relationship to 44AHJD-like phages, which are not known to contain virulence-associated genes. To assess whether these phages might be candidates for applications, in vitro experiments were carried out in which the number of MRSA CC398 cells could be reduced by up to four log₁₀ units. The phages were stable at a wide range of temperatures and pH values.     

Epidemiology and Genotypic Characteristics of Methicillin-Resistant Staphylococcus aureus Strains of Porcine Origin

The main goal of this study was to determine the prevalence of methicillin-resistant Staphylococcus aureus (MRSA), particularly livestock-associated MRSA (LA-MRSA) in pigs and pork. The genotypic relatedness of isolates on the farm, at slaughter, and at the retail level was assessed. Paired nasal and perianal swab samples were collected from 10 cohorts of market-age pigs (24 pigs per cohort) and carcasses at slaughterhouse, and pork samples were collected at retail. Staphylococci were isolated using selective enrichment method. Isolates were tested for antimicrobial resistance by broth microdilution. Duplex PCR was used to confirm MRSA using species-specific (nuc) and methicillin resistance (mecA) genes. The clonal relatedness of isolates was determined using pulsed-field gel electrophoresis (PFGE), Staphylococcus protein A (spa) typing, multilocus sequence typing (MLST), and staphylococcal cassette chromosome mec element (SCCmec) typing. MRSA was detected in 5 of the 10 cohorts (50%), with the prevalence ranging from 0% to 12.5% per cohort. Of all the pigs sampled on the farm before they went to market, 3% (7/240) were MRSA positive. A higher prevalence of MRSA was detected at holding pens at the slaughterhouse (11% [27/240]). MRSA was also detected in 2% (4/235) of the carcasses and 4% (5/135) of the retail pork. While the isolates appear predominantly to be highly clonal, PFGE had a relatively higher discriminatory power (discriminatory index [DI] = 0.624). Four genotypic clusters were identified by PFGE; of the four clusters, clonal type B was predominant across the farm-to-retail continuum. MLST findings revealed that sequence type 5 (ST5) was the most predominant subtype (32/50). The livestock-associated MRSA (clonal complex 398 [CC398] or sequence type 398 [ST398]) was the second common type (12/50) and was detected at all stages from farm to retail. Nine of the 50 (18%) MRSA isolates belonged to spa type 539/t034 that were of ST398 based on MLST. The results of this study confirm that MRSA, including LA-MRSA, is common in herds of swine in Ohio and hereby shown to persist in the farm to processing and retail continuum.     

Epidemiological features,resistance genes,and clones among community-onset methicillin-resistant Staphylococcus aureus (CO-MRSA) isolates detected in northern Spain

Twenty-nine community-onset methicillin-resistant Staphylococcus aureus (CO-MRSA) isolates were prospectively selected according to epidemiological criteria among 374 MRSA isolates collected in our laboratory during 2009–2010 in order to determine which community-associated MRSA (CA-MRSA) and healthcare-associated MRSA (HA-MRSA) clones are circulating in the community in northern Spain. PVL genes were detected in 5 strains (17.2%) that belonged to SCCmec type IV or V and to the agr group I (ST8 and ST2050), agr group II (ST121), and agr group III (ST30 and ST852). These strains were isolated from patients with different clinical manifestations such as urinary tract infection, abscess, or pneumonia, and most of them belonged to emergency department patients with no history of visits to General Practitioners (GPs) in the year before the isolation. We considered that the prevalence of CA-MRSA in community-onset isolates was low (17.2%). A high proportion of the CO-MRSA strains (58.6%) were ST125-MRSA-IVc (CC5), responsible for most of the infections caused by HA-MRSA strains in Spain. This endemic clone is also circulating in the community of northern Spain as we could demonstrate in this study. Antimicrobial resistance was found in spa type t067 isolates linked to the presence of ant(4′)-Ia and msr(A). Most of the CO-MRSA isolates in this study corresponded to spa types more associated to the hospital environment, suggesting the interchange of genetic lineages of MRSA among community and hospital niches.     

Methicillin-resistant Staphylococcus aureus (MRSA) ST398 in a farmer with skin lesions and in pigs of his farm: clonal relationship and detection of lnu(A) gene

Skin infection associated with methicillin-resistant Staphylococcus aureus (MRSA)-ST398 was detected in a pig-farmer, and MRSA-ST398 isolates were also detected in nasal samples of the patient and of 11/12 pigs on his farm. Twelve MRSA isolates were obtained from skin lesions (n = 6) and nasal samples (n = 6) of the patient in two sampling moments and 11 MRSA isolates from nasal samples of pigs. They were typed as t011-SCCmecIVa-agrI and t108-SCCmecV-agrI (patient and pigs) and t588-SCCmecV-agrI (patient). The following resistance genes were detected (number isolates): tet(K) (1), tet(L) (23), tet(M) (13), erm(A) (13), erm(C) (13), msr(A) (11), lnu(A) (21), aph(2″)-acc(6′) (3), ant(4′) (13), aph(3′) (12), dfrS1 (15) and dfrK (22). Seventeen human and animal MRSA-ST398 isolates showed indistinguishable PFGE patterns (A1-spa-t011 or B2-spa-t108) and similar phenotypic-genotypic characteristics, including the presence of the lnu(A) gene, associated with lincomycin resistance. Potential pig-to-human transference of ST398 is suggested in this study. The first detection of the lnu(A) gene in MRSA-ST398 is reported.     

Prevalence and accessory gene regulator (agr) analysis of vancomycin-intermediate Staphylococcus aureus among methicillin-resistant isolates in Taiwan—SMART program, 2003

The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in nosocomial staphylococcal infections in Taiwan has exceeded 50% since 2000. However, little relevant data has been available concerning vancomycin-intermediate S. aureus (VISA) and heteroresistant VISA (hVISA). We collected 1,000 MRSA isolates from ten medical center hospitals in Taiwan during 2003. All were initially screened for reduced susceptibility to vancomycin on brain heart infusion (BHI) agar containing 5 mg/L vancomycin. Among 34 MRSA isolates that grew on the screening plates, two VISA isolates (0.2%) and seven hVISA isolates (0.7%) were evident. Vancomycin-resistant S. aureus was not detected. The accessory gene regulator (agr) typing of all 1,000 MRSA strains were typed by multiplex polymerase chain reaction (PCR); 919 strains (91.9%) including the VISA and hVISA isolates belonged to agr group I, 78 strains (7.8%) were agr group II, two strains (0.2%) were agr group III, and one isolate (0.1%) was agr group IV. There was no relationship between sample sites and agr typing. In 2003, the incidence of hVISA and VISA in Taiwan was low. Continued surveillance is recommended, given the implementation of new Clinical and Laboratory Standards Institute (CLSI) criteria for S. aureus and the increasing clinical use of glycopeptides.     

Characterization of methicillin-resistant Staphylococcus aureus from healthy carrier chickens

Methicillin-resistant Staphylococcus aureus (MRSA) has long been recognized as an important pathogen in human medicine leading to hospital and community-acquired infections. However, it is now also considered a growing problem in veterinary medicine, although causing little or no disease. Although MRSA has already been detected in livestock including poultry, little is known about the epidemiology of MRSA in broiler and layer chickens. We therefore investigated 372 poultry farms in Belgium. We also compared the isolation method recommended by the European Food Safety Authority using two enrichment steps with an isolation method using only one enrichment step. Isolated MRSA was characterized by means of antimicrobial resistance profiling, spa typing, multi-locus sequence typing, and SCCmec typing. MRSA prevalence was 0.8% using the double broth enrichment method, while using the single broth enrichment method it was 1.8%. Five MRSA strains belonged to the livestock-associated (LA) MRSA ST398 (four with spa type t011 and one with t899), and three to the hospital-acquired MRSA ST239 spa type t037. The ST239 strains carried SCCmec type III while those belonging to ST398 carried SCCmec type IV or V. All isolates showed additional resistance to erythromycin and tetracycline apart from the expected resistance to cefoxitin and penicillin. All strains were susceptible to linezolid, mupirocin and vancomycin. In conclusion, a higher sensitivity for the isolation of LA-MRSA was obtained using only one enrichment step. While the typical LA-MRSA ST398 was present at low prevalence in poultry, human-associated strains have also been found.     

Population structure and exotoxin gene content of methicillin-susceptible Staphylococcus aureus from Spanish healthy carriers

The population structure of 111 methicillin-susceptible Staphylococcus aureus (MSSA), recovered in Spain from healthy and risk-free carriers was investigated using pulsed-field gel electrophoresis (PFGE), spa (staphylococcal protein A) typing, multi locus sequence typing (MLST) and the accessory gene regulator (agr). Results from the different techniques were highly concordant, and revealed twelve clonal complexes (CCs): CC30 (27%), CC5 (18.9%), CC45 (16.2%), CC15 (11.7%), CC25 (8.1%), CC1, CC9 (3.6% each), CC59, CC97 and CC121 (2.7% each), CC72 (1.8%) and CC8 (0.9%). Isolates with genetic backgrounds of hospital-acquired MSSA were detected and, consistent with the ability of diverse MSSA to act as recipients of the SCCmec cassette, a MSSA isolate from a healthy carrier shared the ST, spa-type and agr-type of a MRSA clone recovered in a hospital of the same region. All except two fragments of the PGFE-profiles of these isolates were identical, and the differential fragment of the MRSA carried mecA. Analyses of the exotoxin gene content of the nasal isolates revealed an increase in the number of exotoxin genes over time. This, together with the detection of lukPV and the high frequency of tst, exfoliatin and enterotoxin genes, is worrisome and requires further surveillance.     

Epidemiological data of staphylococcal scalded skin syndrome in France from 1997 to 2007 and microbiological characteristics of Staphylococcus aureus associated strains

Epidemiological data on staphylococcal scalded skin syndromes (SSSS), including bullous impetigo (BI) and generalized exfoliative syndrome (GES), are scarce. To better characterize SSSS and associated Staphylococcus aureus strains, we conducted a retrospective study of 349 cases collected in France between 1997 and 2007 by the National Reference Centre of Staphylococci. Our results showed a stationary evolution of SSSS cases, with a heterogeneous distribution of cases in France. Although notification was not exhaustive, we estimated an incidence of 0.56 cases/year/million inhabitants, in accordance with previous studies conducted in France and Europe, with a median age of 2 years old and sex ratios of 1. A seasonal effect was observed, with a higher GES/BI ratio in autumn compared with other seasons, which could be explained by the impact of viral co-infection. Genetic analysis of S. aureus strains showed that accessory gene regulator (agr) 4, exfoliative toxin A (eta) and B (etb) genes, staphylococcal and enterotoxin-like O (selo) gene and agr4 etb selo profiles were predominantly associated with GES, whereas agr2 eta and agr4 eta selo were more frequently observed with BI. Only one methicillin-resistant strain was found. Protein A (spa) typing identified two main genotypes: spa clonal complex (CC) 159/sequence-type (ST) 121 (75%) and spaCC346/ST15 (18%). spaCC159 was mainly associated with agr4 eta etb selo, agr4 eta selo and agr4 etb selo, and spaCC346 was mainly associated with agr2 eta, suggesting that French SSSS cases are caused by these two main lineages. However, in a multivariate analysis, only etb was independently associated with GES.     

Association of biofilm formation and methicillin-resistance with accessory gene regulator (agr) loci in Greek Staphylococcus aureus clones

Pathogenicity of Staphylococcus aureus is coordinated by the accessory gene regulator (agr) system. Previous studies suggested that agr Group II methicillin-resistant S. aureus (MRSA), a polymorphism that has been associated with moderate response to vancomycin, may also be related with overproduction of biofilm. In a hospital environment with endemic occurrence of MRSA, the distribution of agr groups and their association with biofilm formation was investigated. Forty-two MRSA and 32 methicillin-susceptible S. aureus (MSSA) isolates were tested and had derived from 10 genotypes and 8 clonal complexes. agr Groups I, II and IV were evenly distributed among MRSAs and MSSAs but agr Group III was not detected. agr Group II MRSAs showed significantly higher levels of biofilm production in comparison with MRSAs of the remaining agr groups as well as with all three agr groups of MSSAs. These findings suggest that agr Group II is simultaneously associated with methicillin-resistance and biofilm overproduction in a region with endemic MRSA.     

Identification of agr-positive methicillin-resistant Staphylococcus aureus harbouring the class A mec complex by MALDI-TOF mass spectrometry

A small peptide called PSM-mec is encoded on the type II, III and VIII SCCmec cassettes present in the genomes of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) strains. This peptide is excreted by agr-positive strains, which represent about 89% of the strains of our collection and can be identified by the presence of delta toxin in mass spectrometry. The presence of the peptide in the MALDI-TOF MS spectra of whole cells was proved by a knock-down experiment employing a clone that expressed antisense RNA to psm-mec. Furthermore, evaluation of a collection of clinical agr-positive MRSA and MSSA isolates and type strains showed that, using a detection window of m/z 2411–2419, the PSM-mec is detected by mass spectrometry of whole cells with a sensitivity of 0.95 and a specificity of 1, thereby enabling rapid identification of a subgroup of MRSA with a method that is used during routine identification procedures.     

Phenotypic changes of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> during vancomycin therapy for persistent bacteraemia and related clinical outcome

Persistent bacteraemia (PB) due to methicillin-resistant Staphylococcus aureus (MRSA) that fails to respond to glycopeptide therapy is a well-documented clinical problem. There are limited data on changes in agr functionality, vancomycin susceptibility and heteroresistance during MRSA PB. Thus, the frequency of these changes and their clinical significance remain unclear. Only patients with MRSA PB (≥7 days) from a prospective cohort of S. aureus bacteraemia were included. We collected isogenic paired strains and compared vancomycin MIC, vancomycin heteroresistance, and agr functionality between initial and final blood isolates. We also assessed the clinical outcome. A total of 49 patients had MRSA PB over 22 months. Bacteraemia persisted for a median of 13 days and most patients (98%) received glycopeptide as initial therapy. Among 49 isogenic pairs, only one pair showed a vancomycin MIC increase ≥2-fold by broth microdilution method, and only seven (14%) by E-test. Significant portions of initial isolates had vancomycin heteroresistance (49%) and agr dysfunction (76%). Development of vancomycin heteroresistance during PB occurred in four (16%) among 25 initial vancomycin-susceptible isolates, and acquisition of agr dysfunction occurred in two (16%) among 12 initial agr-functional isolates. Changes in the opposite direction occasionally occurred. These phenotypic changes during PB were not associated with mortality, whereas agr dysfunction of the initial isolates was significantly associated with mortality. During MRSA PB, phenotypic changes of MRSA isolates occurred occasionally under prolonged vancomycin exposure but were not significantly associated with clinical outcome. In contrast, initial agr dysfunction could be a predictor for mortality in MRSA PB.     

Endovascular Infections Caused by Methicillin-Resistant Staphylococcus aureus Are Linked to Clonal Complex-Specific Alterations in Binding and Invasion Domains of Fibronectin-Binding Protein A as Well as the Occurrence of fnbB

Endovascular infections caused by Staphylococcus aureus involve interactions with fibronectin present as extracellular matrix or surface ligand on host cells. We examined the expression, structure, and binding activity of the two major S. aureus fibronectin-binding proteins (FnBPA, FnBPB) in 10 distinct, methicillin-resistant clinical isolates from patients with either persistent or resolving bacteremia. The persistent bacteremia isolates (n = 5) formed significantly stronger bonds with immobilized fibronectin as determined by dynamic binding measurements performed with atomic force microscopy. Several notable differences were also observed when the results were grouped by clonal complex 5 (CC5) strains (n = 5) versus CC45 strains (n = 5). Fibronectin-binding receptors on CC5 formed stronger bonds with immobilized fibronectin (P < 0.001). The fnbA gene was expressed at higher levels in CC45, whereas fnbB was found in only CC5 isolates. The fnbB gene was not sequenced because all CC45 isolates lacked this gene. Instead, comparisons were made for fnbA, which was present in all 10 isolates. Sequencing of fnbA revealed discrete differences within high-affinity, fibronectin-binding repeats (FnBRs) of FnBPA that included (i) 5-amino-acid polymorphisms in FnBR-9, FnBR-10, and FnBR-11 involving charged or polar side chains, (ii) an extra, 38-amino-acid repeat inserted between FnBR-9 and FnBR-10 exclusively seen in CC45 isolates, and (iii) CC5 isolates had the SVDFEED epitope in FnBR-11 (a sequence shown to be essential for fibronectin binding), while this sequence was replaced in all CC45 isolates with GIDFVED (a motif known to favor host cell invasion at the cost of reduced fibronectin binding). These complementary sequence and binding data suggest that differences in fnbA and fnbB, particularly polymorphisms and duplications in FnBPA, give S. aureus two distinct advantages in human endovascular infections: (i) FnBPs similar to that of CC5 enhance ligand binding and foster initiation of disease, and (ii) CC45-like FnBPs promote cell invasion, a key attribute in persistent endovascular infections.     

Association of biofilm formation and methicillin-resistance with accessory gene regulator (agr) loci in Greek Staphylococcus aureus clones

Pathogenicity of Staphylococcus aureus is coordinated by the accessory gene regulator (agr) system. Previous studies suggested that agr Group II methicillin-resistant S. aureus (MRSA), a polymorphism that has been associated with moderate response to vancomycin, may also be related with overproduction of biofilm. In a hospital environment with endemic occurrence of MRSA, the distribution of agr groups and their association with biofilm formation was investigated. Forty-two MRSA and 32 methicillin-susceptible S. aureus (MSSA) isolates were tested and had derived from 10 genotypes and 8 clonal complexes. agr Groups I, II and IV were evenly distributed among MRSAs and MSSAs but agr Group III was not detected. agr Group II MRSAs showed significantly higher levels of biofilm production in comparison with MRSAs of the remaining agr groups as well as with all three agr groups of MSSAs. These findings suggest that agr Group II is simultaneously associated with methicillin-resistance and biofilm overproduction in a region with endemic MRSA.