Clonal Complex 398 Methicillin Susceptible Staphylococcus aureus: A Frequent Unspecialized Human Pathogen with Specific Phenotypic and Genotypic Characteristics

Clonal complex 398 livestok-associated-MRSA (CC398 LA-MRSA) clone is described as a major animal pathogen that can also colonize and infect humans. CC398 methicillin susceptible Staphylococcus aureus (CC398 MSSA) is less described. We identified 126 CC398 MSSA strains of human origin within 6380 S. aureus isolates gathered between 2009 and 2011, from the French National Reference Centre for Staphylococci. They were characterized using antimicrobial susceptibility testing, spa typing, DNA microarrays (Identibac S. aureus Genotyping ®, Alere), CC398-specific sequence PCR, ermT (encoding macrolides résistance) PCR. Fifty-three CC398 LA-MRSA collected from French pigs and veal were used as comparators, and phylogenetic relations between human CC398 MSSA and animal CC398 MRSA populations were explored on the basis of spa-typing and DNA microarrays. CC398 MSSA were able to induce a large spectrum of infections (especially skin, bloodstream, and pneumonias). The prevalence rate of this clone was high in MSSA population, i.e., 24.7% in a local prospective study on nasal colonization, and 7.5% in a national prospective study on infective endocarditis. CC398 MSSA isolates were frequently (89%) erythromycin resistant, due to the presence of the ermT gene, a gene not detected in erythromycin resistant CC398 LA-MRSA strains. Expression of staphylococcal complement inhibitor (scn) and the chemotaxis inhibitory protein (chp), was also specific to this population. The CC398 MRSA signature included also a panel of antibiotic resistance genes, especially a type IV or V cassette mec and tetM. CC398 MSSA and CC398 LA-MRSA populations were closely related based on spa-typing and DNA microarrays, with the MRSA strains forming the most derived lineage in phylogenic trees. Both MSSA and MRSA populations may come from common ancestors, which would have evolved in the settings of different selective pressures, explaining the acquisition of ermT, chp and scn for MSSA, and antibiotic resistance genes for MRSA.     

Livestock-Associated Methicillin-Resistant Staphylococcus aureus (MRSA) as Causes of Human Infection and Colonization in Germany

Pigs, cattle and poultry are colonized with MRSA and the zoonotic transmission of such MRSA to humans via direct animal contact, environmental contaminations or meat are a matter of concern. Livestock-associated (LA) MRSA are mostly belonging to clonal complex (CC) 398 as defined by multilocus sequence typing. However, MRSA of other clonal lineages including CC5, CC9 and CC97 have also been detected in livestock animals in Germany. Within the framework of a Dutch-German network project (EUREGIO), 14,036 MRSA isolated from clinical and screening specimens (January 2008 - June 2012) derived from human patients in hospitals as well as general or specialized practices in a German region characterized by a high density of livestock production, were subjected to S. aureus protein A (spa) sequence typing. The prevalence of putative LA-MRSA among the human MRSA isolates was determined by analyzing the detection of livestock-indicator (LI) spa types which had already been reported in German livestock. Overall, 578 spa types were detected among the MRSA isolates. LI spa types t011, t034, t108, t1451, t2011, t571, t1456, t1250, t1255, t1580, t2970, t2346, t1344, t2576, t2330 and t2510 (all of which are indicative for LA-MRSA CC398) accounted for 18.6% of all human isolates. The LI spa types t1430 (CC9), t3992 (CC97), t002 (CC5) and t007 (CC30) were found in 0.14%, 0.01%, 1.01% and 0.04% of all human MRSA isolates, respectively. LI spa types associated with CC398 represented 23% of all MRSA from screening samples and a varying proportion among isolates from clinical specimens ranging between 0% in cerebrospinal fluid, 8% in blood cultures and 14% in deep respiratory fluids. Our findings indicate that LA-MRSA are a major cause for human infection and stress the need for close surveillance. Although LA-MRSA CC398 predominates, the occurrence of putative LA-MRSA from other clonal lineages should be monitored.     

A Livestock-Associated,Multidrug-Resistant,Methicillin-Resistant Staphylococcus aureus Clonal Complex 97 Lineage Spreading in Dairy Cattle and Pigs in Italy

Pandemic methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 97 (CC97) lineages originated from livestock-to-human host jumps. In recent years, CC97 has become one of the major MRSA lineages detected in Italian farmed animals. The aim of this study was to characterize and analyze differences in MRSA and methicillin-susceptible S. aureus (MSSA) mainly of swine and bovine origins. Forty-seven CC97 isolates, 35 MRSA isolates, and 6 MSSA isolates from different Italian pig and cattle holdings; 5 pig MRSA isolates from Germany; and 1 human MSSA isolate from Spain were characterized by macrorestriction pulsed-field gel electrophoresis (PFGE) analysis, multilocus sequence typing (MLST), spa typing, staphylococcal cassette chromosome mec (SCCmec) typing, and antimicrobial resistance pattern analysis. Virulence and resistance genes were investigated by PCR and microarray analysis. Most of the isolates were of SCCmec type V (SCCmec V), except for two German MRSA isolates (SCCmec III). Five main clusters were identified by PFGE, with the German isolates (clusters I and II) showing 60.5% similarity with the Italian isolates, most of which (68.1%) grouped into cluster V. All CC97 isolates were Panton-Valentine leukocidin (PVL) negative, and a few (n = 7) tested positive for sak or scn. All MRSA isolates were multidrug resistant (MDR), and the main features were erm(B)- or erm(C)-mediated (n = 18) macrolide-lincosamide-streptogramin B resistance, vga(A)-mediated (n = 37) pleuromutilin resistance, fluoroquinolone resistance (n = 33), tet(K) in 32/37 tet(M)-positive isolates, and blaZ in almost all MRSA isolates. Few host-associated differences were detected among CC97 MRSA isolates: their extensive MDR nature in both pigs and dairy cattle may be a consequence of a spillback from pigs of a MRSA lineage that originated in cattle as MSSA and needs further investigation. Measures should be implemented at the farm level to prevent spillover to humans in intensive farming areas.     

Alarming Proportions of Methicillin-Resistant Staphylococcus aureus (MRSA) in Wound Samples from Companion Animals,Germany 2010–2012

Staphylococcus (S.) aureus is an important cause of wound infections in companion animals, and infections with methicillin-resistant S. aureus (MRSA) are of particular concern due to limited treatment options and their zoonotic potential. However, comparable epidemiological data on MRSA infections in dogs, cats and horses is scarce, also limiting the knowledge about possible links to MRSA isolates from human populations. To gain more knowledge about the occurrence and genotypic variation of MRSA among wound swabs of companion animal origin in Germany we performed a survey (2010–2012) including 5,229 samples from 1,170 veterinary practices. S. aureus was identified in 201 (5.8%) canine, 140 (12.2%) feline and 138 (22.8%) equine swabs from a total of 3,479 canine, 1,146 feline and 604 equine wounds, respectively. High MRSA rates were identified with 62.7%, 46.4% and 41.3% in S. aureus of canine, feline and equine origin, respectively. Further genotyping including spa typing and multilocus sequence typing (MLST) revealed a comparable distribution of spa types among canine and feline MRSA with CC22 (47.6%; 49.2%) and CC5 (30.2%; 29.2%) as predominant lineages followed by CC398 (13.5%; 7.7%) and CC8 (4.0%; 9.2%). In contrast, the majority of equine MRSA belonged to CC398 (87.7%). Our data highlight the importance of S. aureus and MRSA as a cause of wound infections, particularly in cats and horses in Germany. While “human-associated” MRSA lineages were most common in dogs and cats, a remarkable number of CC398-MRSA was detected in horses, indicating a replacement of CC8-MRSA as the predominant lineage within horses in Germany. These data enforce further longitudinal epidemiological approaches to examine the diversity and temporal relatedness of MRSA populations in humans and animals to assess probable sources of MRSA infections. This would enable a sound risk assessment and establishment of intervention strategies to limit the additional spread of MRSA.     

Molecular characterization of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates from various healthcare institutions in Nairobi,Kenya: a cross sectional study

Background

Staphylococcus aureus (S. aureus) has established itself over the years as a major cause of morbidity and mortality both within the community and in healthcare settings. Methicillin resistant S. aureus (MRSA) in particular has been a major cause of nosocomial infections resulting in significant increase in healthcare costs. In Africa, the MRSA prevalence has been shown to vary across different countries. In order to better understand the epidemiology of MRSA in a setting, it is important to define its population structure using molecular tools as different clones have been found to predominate in certain geographical locations.

Methods

We carried out PFGE, MLST, SCCmec and spa typing of selected S. aureus isolates from a private and public referral hospital in Nairobi, Kenya.

Results

A total of 93 S. aureus isolates were grouped into 19 PFGE clonal complexes (A–S) and 12 singletons. From these, 55 (32 MRSA and 23 MSSA) representative isolates from each PFGE clonal complex and all singletons were spa typed. There were 18 different MRSA spa types and 22 MSSA spa types. The predominant MRSA spa type was t037 comprising 40.6 % (13/32) of all MRSA. In contrast, the MSSA were quite heterogeneous, only 2 out of 23 MSSA shared the same spa type. Two new MRSA spa types (t13149 and t13150) and 3 new MSSA spa types (t13182, t13193 and t13194) were identified. The predominant clonal complex was CC 5 which included multi-locus sequence types 1, 8 and 241.

Conclusion

In contrast to previous studies published from Kenya, there’s marked genetic diversity amongst clinical MRSA isolates in Nairobi including the presence of well-known epidemic MRSA clones. Given that these clones are resident within our referral hospitals, adherence to strict infection control measures needs to be ensured to reduce morbidity and mortality associated with hospital acquired MRSA infections.

     

Methicillin Susceptible Staphylococcus aureus (MSSA) of Clonal Complex CC398, t571 from Infections in Humans Are Still Rare in Germany

Methicillin-susceptible Staphylococcus aureus (MSSA) attributed to clonal complex (CC) 398 and exhibiting spa-type t571 received attention in Europe and in the USA for being associated with severe infections in humans. As this spa-type is exhibited by livestock-associated (LA) Methicillin-resistant S. aureus (MRSA) as well, it is important to discriminate LA- and human-derived strains by easy to perform, PCR-based methods. MSSA t571 contain phage int3 carrying scn and chp, whereas LA-MRSA t571 lack these markers. In contrast, pathogenicity island SaPIbov5 (detected by PCR bridging vwbbov and scn) is contained by LA-MRSA t571 and absent in the human MSSA subpopulation. Furthermore, MSSA t571 contain erm(T), the particular genomic arrangement of which was assessed by a PCR bridging erm(T) and the adjacent transposase gene. MSSA t571 are rare so far in Germany among isolates from infections in humans (0.14%) as well as among isolates from nasal colonization (0.13%). LA-MRSA t571 are also infrequent among MRSA isolated from carriage at admission to hospitals (0.1%) and also among isolates from infections in humans (0.013%).     

Characterization of Staphylococcus aureus from Distinct Geographic Locations in China: An Increasing Prevalence of spa-t030 and SCCmec Type III

Staphylococcus aureus belongs to one of the most common bacteria causing healthcare and community associated infections in China, but their molecular characterization has not been well studied. From May 2011 to June 2012, a total of 322 non-duplicate S. aureus isolates were consecutively collected from seven tertiary care hospitals in seven cities with distinct geographical locations in China, including 171 methicillin sensitive S. aureus (MSSA) and 151 MRSA isolates. All isolates were characterized by spa typing. The presence of virulence genes was tested by PCR. MRSA were further characterized by SCCmec typing. Seventy four and 16 spa types were identified among 168 MSSA and 150 MRSA, respectively. One spa type t030 accounted for 80.1% of all MRSA isolates, which was higher than previously reported, while spa-t037 accounted for only 4.0% of all MRSA isolates. The first six spa types (t309, t189, t034, t377, t078 and t091) accounted for about one third of all MSSA isolates. 121 of 151 MRSA isolates (80.1%) were identified as SCCmec type III. pvl gene was found in 32 MSSA (18.7%) and 5 MRSA (3.3%) isolates, with ST22-MSSA-t309 as the most commonly identified strain. Compared with non-epidemic MRSA clones, epidemic MRSA clones (corresponding to ST239) exhibited a lower susceptibility to rifampin, ciprofloxacin, gentamicin and trimethoprim-sulfamethoxazole, a higher prevalence of sea gene and a lower prevalence of seb, sec, seg, sei and tst genes. The increasing prevalence of multidrug resistant spa-t030 MRSA represents a major public health problem in China.     

Geographic Distribution of Staphylococcus aureus Causing Invasive Infections in Europe: A Molecular-Epidemiological Analysis

Background

Staphylococcus aureus is one of the most important human pathogens and methicillin-resistant variants (MRSAs) are a major cause of hospital and community-acquired infection. We aimed to map the geographic distribution of the dominant clones that cause invasive infections in Europe.

Methods and Findings

In each country, staphylococcal reference laboratories secured the participation of a sufficient number of hospital laboratories to achieve national geo-demographic representation. Participating laboratories collected successive methicillin-susceptible (MSSA) and MRSA isolates from patients with invasive S. aureus infection using an agreed protocol. All isolates were sent to the respective national reference laboratories and characterised by quality-controlled sequence typing of the variable region of the staphylococcal spa gene (spa typing), and data were uploaded to a central database. Relevant genetic and phenotypic information was assembled for interactive interrogation by a purpose-built Web-based mapping application. Between September 2006 and February 2007, 357 laboratories serving 450 hospitals in 26 countries collected 2,890 MSSA and MRSA isolates from patients with invasive S. aureus infection. A wide geographical distribution of spa types was found with some prevalent in all European countries. MSSA were more diverse than MRSA. Genetic diversity of MRSA differed considerably between countries with dominant MRSA spa types forming distinctive geographical clusters. We provide evidence that a network approach consisting of decentralised typing and visualisation of aggregated data using an interactive mapping tool can provide important information on the dynamics of MRSA populations such as early signalling of emerging strains, cross border spread, and importation by travel.

Conclusions

In contrast to MSSA, MRSA spa types have a predominantly regional distribution in Europe. This finding is indicative of the selection and spread of a limited number of clones within health care networks, suggesting that control efforts aimed at interrupting the spread within and between health care institutions may not only be feasible but ultimately successful and should therefore be strongly encouraged. Please see later in the article for the Editors'' Summary     

Matched-Cohort DNA Microarray Diversity Analysis of Methicillin Sensitive and Methicillin Resistant Staphylococcus aureus Isolates from Hospital Admission Patients

As genotyping of S. aureus is important for epidemiologic research and for hygiene management, methods are required for standardized fast and easily applicable evaluation of closely related epidemic strains with high prevalence in hospitals. In this single centre matched control study we compared a new commercially available DNA microarray (IdentiBAC) with standard spa-typing for S. aureus genotyping. Included in the study was a subgroup of 46 MRSA and matched 46 MSSA nasal isolates of the Saarland University Medical Center collected during a state-wide admission prevalence screening. Microarray (MA) and also spa-typing could easily differentiate the genetically diverse MSSA group. However, due to the predominance of CC5/t003 in the MRSA group a sufficient subtyping required analysis of more complex genetic profiles as was shown here by the MA comprising a total number of 334 different hybridization probes. The genetic repertoire of the MRSA group was characterized by more virulence genes as compared to the MSSA group. The standard evaluation of MA results by the original software into CCs, agr-, SCCmec- and capsule-types was substituted in the present study by implementation of multivariate subtyping of closely related CC5 isolates using three different bioinformatic methods (splits graph, cluster dendrogram, and principal component analysis). Each method used was applicable for standardized and highly discriminative subtyping with high concordance. We propose that the identified S. aureus subtypes with characteristic virulence gene profiles are presumably associated also with virulence and pathogenicity in vivo; however, this remains to be analyzed in future studies. MA was superior to spa-typing for epidemiologic and presumably also provide functional respectively virulence associated characterization of S. aureus isolates. This is of specific importance for the hospital setting. In future, MA could become a new standard test for S. aureus typing in combination with multivariate bioinformatic analysis.     

Molecular Typing of MRSA and of Clinical Staphylococcus aureus Isolates from Ia?i,Romania

Romania is one of the countries with the highest prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in the world. To obtain data on affiliation of MRSA to strains and clonal complexes and on the population of methicillin susceptible S. aureus (MSSA), clinical isolates from bloodstream infections, skin and soft tissue infections as well as from screening swabs were collected at hospitals in Ia?i, a city in the North-Eastern part of Romania. Isolates were characterised by microarray hybridisation. Nearly half of all isolates (47%), and about one third (34%) of bloodstream isolates were MRSA. The prevalence of the Panton-Valentine leukocidin (PVL) was also high (31% among MRSA, 14% among MSSA). The most common MRSA strain was a PVL-negative CC1-MRSA-IV that might have emerged locally, as a related MSSA was also common. PVL-positive CC8-MRSA-IV (“USA300”) and PVL-negative ST239-like MRSA-III were also frequently found while other MRSA strains were only sporadically detected. Among MSSA, PVL-positive CC121 as well as PVL-negative CC1, CC22 and CC45 predominated. Although this study provides only a snapshot of S. aureus/MRSA epidemiology in Romania, it confirms the high burden of MRSA and PVL on Romanian healthcare settings.     

MRSA Clonal Complex 22 Strains Harboring Toxic Shock Syndrome Toxin (TSST-1) Are Endemic in the Primary Hospital in Gaza,Palestine

Background

Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen in both community and healthcare-related settings worldwide. Current knowledge regarding the epidemiology of S. aureus and MRSA in Gaza is based on a single community-based carriage study. Here we describe a cross-sectional analysis of 215 clinical isolates collected from Al-Shifa Hospital in Gaza during 2008 and 2012.

Methods

All isolates were characterized by spa typing, SCCmec typing, and detection of genes encoding Panton-Valentine leukocidin (PVL) and toxic shock syndrome toxin (TSST-1). Representative genotypes were also subjected to multilocus sequence typing (MLST). Antibiotic susceptibility testing was performed using VITEK2 and MicroScan.

Results

MRSA represented 56.3% of all S. aureus strains, and increased in frequency from 2008 (54.8%) to 2012 (58.4%). Aside from beta-lactams, resistance was observed to tetracycline, erythromycin, clindamycin, gentamicin, and fluoroquinolones. Molecular typing identified 35 spa types representing 17 MLST clonal complexes (CC), with spa 998 (Ridom t223, CC22) and spa 70 (Ridom t044, CC80) being the most prevalent. SCCmec types I, III, IV, V and VI were identified among MRSA isolates, while type II was not detected. PVL genes (lukF/S-PV) were detected in 40.0% of all isolates, while the TSST-1 gene (tst) was detected in 27.4% of all isolates, with surprisingly high frequency within CC22 (70.4%). Both PVL and TSST-1 genes were found in several isolates from 2012.

Conclusions

Molecular typing of clinical isolates from Gaza hospitals revealed unusually high prevalence of TSST-1 genes among CC22 MRSA, which is noteworthy given a recent community study describing widespread carriage of a CC22 MRSA clone known as the ‘Gaza strain’. While the latter did not address TSST-1, tst-positive spa 998 (Ridom t223) has been detected in several neighboring countries, and described as endemic in an Italian NICU, suggesting international spread of a ‘Middle Eastern variant’ of pandemic CC22 strain EMRSA-15.     

Detection of Methicillin-Susceptible Staphylococcus aureus ST398 and ST133 Strains in Gut Microbiota of Healthy Humans in Spain

Fecal samples of 100 healthy humans were tested for Staphylococcus aureus recovery. Fifteen samples (15 %) contained S. aureus, all methicillin-susceptible (MSSA), being one isolate/sample further studied. These 15 isolates were characterized by spa and agr typing as well as multi-locus sequence typing. High diversity of spa types (n?=?11) and sequences types (n?=?8) was detected. Two S. aureus of lineages ST398 or ST133 were detected, and six isolates were ascribed to clonal complex 30 (CC30). Strains were susceptible to most of the 17 antimicrobial agents tested with exceptions: erythromycin/clindamycin (three strains, containing erm(C) and/or erm(A) + mph(C) genes) and tobramycin and mupirocin (one strain containing ant(4′)-Ia + mup(A) genes). The presence of 18 staphylococcal enterotoxin genes was studied by PCR, and isolates were negative for lukF/lukS-PV genes, although strain ST133 harbored the lukD-lukE + lukM genes. Other virulence genes detected were (number of strains): tsst-1 (6), hla (15), hlb (9), hld (15), hlg (6), hlgv (9), cna (2), aur (14), and egc-like cluster (3). Analysis of immune evasion cluster genes showed six types, highlighting their absence in two strains of lineages ST133 and ST5. A high clonal diversity of MSSA strains was identified in the intestinal microbiota of healthy humans, being CC30 the most frequent one. This is the first report of MSSA ST133 and ST398 isolates in gut microbiota of healthy humans.     

Evaluation of Multiple-Locus Variable Number of Tandem Repeats Analysis for Typing Livestock-Associated Methicillin-Resistant Staphylococcus aureus

Background

The increasing occurrence of livestock-associated (LA) methicillin-resistant Staphylococcus aureus (MRSA) associated with the clonal complex (CC) 398 within the past years shows the importance of standardized and comparable typing methods for the purposes of molecular surveillance and outbreak detection. Multiple-locus variable number of tandem repeats analysis (MLVA) has recently been described as an alternative and highly discriminative tool for S. aureus. However, until now the applicability of MLVA for the typing of LA-MRSA isolates from different geographic origin has not been investigated in detail. We therefore compared MLVA and S. aureus protein A (spa) typing for characterizing porcine MRSA from distinct Dutch and German farms.

Methodology/Principal Findings

Overall, 134 MRSA isolates originating from 21 different pig-farms in the Netherlands and 36 farms in Germany comprising 21 different spa types were subjected to MLVA-typing. Amplification and subsequent automated fragment sizing of the tandem repeat loci on a capillary sequencer differentiated these 134 isolates into 20 distinct MLVA types. Whereas overall MLVA and spa typing showed the same discriminatory power to type LA-MRSA (p  = 0.102), MLVA was more discriminatory than spa typing for isolates associated with the prevalent spa types t011 and t034 (Simpson’s Index of Diversity 0.564 vs. 0.429, respectively; p<0.001).

Conclusion

Although the applied MLVA scheme was not more discriminatory than spa typing in general, it added valuable information to spa typing results for specific spa types (t011, t034) which are highly prevalent in the study area, i.e. Dutch-German border area. Thus, both methods may complement each other to increase the discriminatory power to resolute highly conserved clones such as CC398 (spa types t011, t034) for the detection of outbreaks and molecular surveillance of zoonotic MRSA.     

Subpopulations of Staphylococcus aureus Clonal Complex 121 Are Associated with Distinct Clinical Entities

We investigated the population structure of Staphylococcus aureus clonal complex CC121 by mutation discovery at 115 genetic housekeeping loci from each of 154 isolates, sampled on five continents between 1953 and 2009. In addition, we pyro-sequenced the genomes from ten representative isolates. The genome-wide SNPs that were ascertained revealed the evolutionary history of CC121, indicating at least six major clades (A to F) within the clonal complex and dating its most recent common ancestor to the pre-antibiotic era. The toxin gene complement of CC121 isolates was correlated with their SNP-based phylogeny. Moreover, we found a highly significant association of clinical phenotypes with phylogenetic affiliations, which is unusual for S. aureus. All isolates evidently sampled from superficial infections (including staphylococcal scalded skin syndrome, bullous impetigo, exfoliative dermatitis, conjunctivitis) clustered in clade F, which included the European epidemic fusidic-acid resistant impetigo clone (EEFIC). In comparison, isolates from deep-seated infections (abscess, furuncle, pyomyositis, necrotizing pneumonia) were disseminated in several clades, but not in clade F. Our results demonstrate that phylogenetic lineages with distinct clinical properties exist within an S. aureus clonal complex, and that SNPs serve as powerful discriminatory markers, able to identify these lineages. All CC121 genomes harboured a 41-kilobase prophage that was dissimilar to S. aureus phages sequenced previously. Community-associated MRSA and MSSA from Cambodia were extremely closely related, suggesting this MRSA arose in the region.     

Livestock-Associated Methicillin Resistant and Methicillin Susceptible Staphylococcus aureus Sequence Type (CC)1 in European Farmed Animals: High Genetic Relatedness of Isolates from Italian Cattle Herds and Humans

Methicillin-resistant Staphylococcus aureus (MRSA) Sequence Type (ST)1, Clonal Complex(CC)1, SCCmec V is one of the major Livestock-Associated (LA-) lineages in pig farming industry in Italy and is associated with pigs in other European countries. Recently, it has been increasingly detected in Italian dairy cattle herds. The aim of this study was to analyse the differences between ST1 MRSA and methicillin-susceptible S. aureus (MSSA) from cattle and pig herds in Italy and Europe and human isolates. Sixty-tree animal isolates from different holdings and 20 human isolates were characterized by pulsed-field gel electrophoresis (PFGE), spa-typing, SCCmec typing, and by micro-array analysis for several virulence, antimicrobial resistance, and strain/host-specific marker genes. Three major PFGE clusters were detected. The bovine isolates shared a high (≥90% to 100%) similarity with human isolates and carried the same SCCmec type IVa. They often showed genetic features typical of human adaptation or present in human-associated CC1: Immune evasion cluster (IEC) genes sak and scn, or sea; sat and aphA3-mediated aminoglycoside resistance. Contrary, typical markers of porcine origin in Italy and Spain, like erm(A) mediated macrolide-lincosamide-streptograminB, and of vga(A)-mediated pleuromutilin resistance were always absent in human and bovine isolates. Most of ST(CC)1 MRSA from dairy cattle were multidrug-resistant and contained virulence and immunomodulatory genes associated with full capability of colonizing humans. As such, these strains may represent a greater human hazard than the porcine strains. The zoonotic capacity of CC1 LA-MRSA from livestock must be taken seriously and measures should be implemented at farm-level to prevent spill-over.     

Spa typing of Staphylococcus aureus Isolated from Clinical Specimens from Outpatients in Iraq

Methicillin-resistant Staphylococcus aureus (MRSA) is notorious as a hospital superbug and a problematic pathogen among communities. The incidence of MRSA has substantially increased over time in Iraq. The aim of this study was to determine the prevalence and spa types of MRSA isolates from outpatients or patients upon admission into hospitals. Various biochemical tests identified S. aureus isolates, and then this identification was confirmed by PCR using species-specific 16S rRNA primer pairs. Antibiotic susceptibility was determined against methicillin, oxacillin, and vancomycin using the disk diffusion method. Vancomycin MIC was detected by VITEK 2 compact system. All the identified isolates were screened for the presence of mecA and lukS-PV-lukF-PV genes; 36 of them were subjected to spa typing-based PCR. Out of 290 clinical samples, 65 (22.4%) were S. aureus, of which 62 (95.4%) strains were resistant to oxacillin and methicillin. Except for two isolates, all MRSA isolates were mecA positive. One of the three MSSA isolates was mecA positive. Five strains were resistant to vancomycin. Fourteen (21.5%) isolates were positive for the presence of lukS-PV-lukF-PV genes. Spa typing of 36 S. aureus isolates revealed eleven different spa types, t304 (30.3%), t307 (19.4%), t346 (8.3%), t044 (8.3%), t15595 (8.3%), t386 (5.5%), t5475 (5.5%), t17928 (2.8%), t14870 (2.8%), t021 (2.8%), and t024 (2.8%). These findings could be useful for assessing the genetic relatedness of strains in the region for epidemiological and monitoring purposes, which would be essential to limiting the spread of MRSA.     

Clonal distribution of bone sialoprotein-binding protein gene among Staphylococcus aureus isolates associated with bloodstream infections

Staphylococcus aureus is a leading cause of bloodstream infections (BSI) and diseases that may be caused by hematogenous spread. The staphylococcal adhesin, for which the association with the infections emerging as a complication of septicemia has been well documented, is a bone sialoprotein-binding protein (Bbp). The aim of the study was to assess the prevalence of a bbp gene in S. aureus bloodstream isolates associated with BSI and to investigate to what degree the distribution of this gene is linked to the clonality of the population. Spa typing, used in order to explore the genetic population structure of the isolates, yielded 29 types. Six spa clusters and seven singletons were identified. The most frequent was spa clonal complex CC021 associated with MLST CC30 (38 %). The bbp gene was found in 47 % of isolates. Almost all isolates (95 %) clustered in spa clonal complex CC021 were positive for this gene. All isolates carrying the bbp gene were sensitive to methicillin, and if clustered in the spa CC021, belonged to agr group III. Our study shows that Bbp is not strictly associated with BSI. However, one may conclude that for clonally related S. aureus strains most commonly causing BSI, the risk of Bbp-mediated complications of septicemia is expected to be higher than for other strains.     

Molecular Characterization of Staphylococcus aureus from Patients with Surgical Site Infections at Mulago Hospital in Kampala,Uganda

Background

The prevalence of Methicillin resistant Staphylococcus aureus (MRSA) is progressively increasing globally with significant regional variation. Understanding the Staphylococcus aureus lineages is crucial in controlling nosocomial infections. Recent studies on S. aureus in Uganda have revealed an escalating burden of MRSA. However, the S. aureus genotypes circulating among patients are not known. Here, we report S. aureus lineages circulating in patients with surgical site infections (SSI) at Mulago National hospital, Kampala, Uganda.

Methods

A cross-sectional study involving 314 patients with SSI at Mulago National Hospital was conducted from September 2011 to April 2012. Pus swabs from the patients’ SSI were processed using standard microbiological procedures. Methicillin sensitive Staphylococcus aureus (MSSA) and MRSA were identified using phenotypic tests and confirmed by PCR-detection of the nuc and mecA genes, respectively. SCCmec genotypes were determined among MRSA isolates using multiplex PCR. Furthermore, to determine lineages, spa sequence based-genotyping was performed on all S. aureus isolates.

Results

Of the 314 patients with SSI, S. aureus accounted for 20.4% (64/314), of which 37.5% (24/64) were MRSA. The predominant SCCmec types were type V (33.3%, 8/24) and type I (16.7%, 4/24). The predominant spa lineages were t645 (17.2%, 11/64) and t4353 (15.6%, 10/64), and these were found to be clonally circulating in all the surgical wards. On the other hand, lineages t064, t355, and t4609 were confined to the obstetrics and gynecology wards. A new spa type (t10277) was identified from MSSA isolate. On multivariate logistic regression analysis, cancer and inducible clindamycin resistance remained as independent predictors of MRSA-SSI.

Conclusion

SCCmec types I and V are the most prevalent MRSA mecA types from the patients’ SSI. The predominant spa lineages (t645 and t4353) are clonally circulating in all the surgical wards, calling for strengthening of infection control practices at Mulago National Hospital.     

Antimicrobial Resistance of Staphylococcus aureus Strains Acquired by Pig Farmers from Pigs

Carriage of animal-associated methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 398 (CC398) is common among pig farmers. This study was conducted (i) to investigate whether pig farmers are colonized with pig-specific S. aureus genotypes other than CC398 and (ii) to survey antimicrobial resistance of S. aureus isolates from pigs and pig farmers. Forty-eight S. aureus isolates from pig farmers and veterinarians and 130 isolates from pigs collected in Western Switzerland were genotyped by spa typing and amplified fragment length polymorphism (AFLP). Antimicrobial resistance profiles were determined for representative sample of the isolates. The data obtained earlier on healthy S. aureus carriers without exposure to agriculture were used for comparison. The genotype composition of S. aureus isolates from pig farmers and veterinarians was similar to isolates from pigs with predominant AFLP clusters CC398, CC9, and CC49. The resistance to tetracycline and macrolides (clarithromycin) was common among the isolates from farmers and veterinarians (52 and 21%, respectively) and similar to resistance levels in isolates from pigs (39 and 23%, respectively). This was in contrast to isolates from persons without contact with agriculture, where no (0/128) isolates were resistant to tetracycline and 3% of the isolates were resistant to clarithromycin. MRSA CC398 was isolated from pigs (n = 11) and pig farmers (n = 5). These data imply that zoonotic transmission of multidrug-resistant S. aureus from pigs to farmers is frequent, and well-known MRSA transmission merely represents the tip of the iceberg for this phenomenon. We speculate that the relatively low frequency of MRSA isolation is related to lower antimicrobial use in Switzerland compared to, for example, the Netherlands.     

The Emergence and Spread of Multiple Livestock-Associated Clonal Complex 398 Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Strains among Animals and Humans in the Republic of Ireland, 2010–2014

Clonal complex (CC) 398 methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) are associated with carriage and infection among animals and humans but only a single case of CC398 MRSA has been reported in the Republic of Ireland (ROI). The present study investigated the molecular epidemiology of CC398 MRSA (n = 22) and MSSA (n = 10) from animals and humans in the ROI from 2010–2014. Isolates underwent antimicrobial susceptibility testing, spa typing, DNA microarray profiling and PCR for CC398-associated resistance genes. All MRSA underwent SCCmec IV or V subtyping. Four distinct CC398-MRSA incidents were identified from (i) a man in a nursing home (spa type t011-SCCmec IVa, immune evasion complex (IEC) negative), (ii) a horse and veterinarian who had recently travelled to Belgium (t011-IVa, IEC positive), (iii) pigs (n = 9) and farm workers (n = 9) on two farms, one which had been restocked with German gilts and the other which was a finisher farm (t034-V_T, IEC negative, 3/9 pigs; t011- V_T, IEC negative, 6/9 pigs & 9/9 farm workers), and (iv) a child who had worked on a pig farm in the UK (t034-V_T, IEC negative). Isolates also carried different combinations of multiple resistance genes including erm(A), erm(B), tet(K), tet(M) & tet(L), fexA, spc, dfrG, dfrK aacA-aphD and aadD further highlighting the presence of multiple CC398-MRSA strains. CC398 MSSA were recovered from pigs (n = 8) and humans (n = 2). CC398 MSSA transmission was identified among pigs but zoonotic transmission was not detected with animal and human isolates exhibiting clade-specific traits. This study highlights the importation and zoonotic spread of CC398 MRSA in the ROI and the spread of CC398 MSSA among pigs. Increased surveillance is warranted to prevent further CC398 MRSA importation and spread in a country that was considered CC398 MRSA free.