Characterization of SCCmec Instability in Methicillin-Resistant Staphylococcus aureus Affecting Adjacent Chromosomal Regions, Including the Gene for Staphylococcal Protein A (spa).

Staphylococcal cassette chromosome mec (SCCmec) represents a sequence of clear clinical and diagnostic importance in staphylococci. At a minimum the chromosomal cassette contains the mecA gene encoding PBP2a but frequently also includes additional antibiotic resistance genes (e.g., ermA and aadC; macrolide and aminoglycoside resistance, respectively). Certain regions within SCCmec elements are hot spots for sequence instability due to cassette-specific recombinases and a variety of internal mobile elements. SCCmec changes may affect not only cassette stability but the integrity of adjacent chromosomal sequences (e.g., the staphylococcal protein A gene; spa). We investigated SCCmec stability in methicillin-resistant Staphylococcus aureus (MRSA) strains carrying one of four SCCmec types cultured in the absence of antimicrobial selection over a 3-month period. SCCmec rearrangements were first detected in cefoxitin-susceptible variants after 2 months of passage, and most commonly showed precise excision of the SCCmec element. Sequence analysis after 3 months revealed both precise SCCmec excision and a variety of SCCmec internal deletions, some including extensive adjacent chromosomal loss, including spa. No empty cassettes (i.e., loss of just mecA from SCCmec) were observed among the variants. SCCmec stability was influenced both by internal mobile elements (IS431) as well as the host cell environment. Genotypically similar clinical isolates with deletions in the spa gene were also included for purposes of comparison. The results indicate a role for host-cell influence and the IS431 element on SCCmec stability.

Presence of Clostridioides difficile and multidrug-resistant healthcare-associated pathogens in stool specimens from hospitalized patients in the USA.

BACKGROUND: Healthcare-associated infections (HCAIs) continue to be a major cause of morbidity and mortality. Many HCAI pathogens, including multidrug-resistant organisms (MDROs), colonize the gastrointestinal tract. AIM: To determine the frequency of MDRO carriage in patients who do and do not harbour toxigenic Clostridioides difficile in their stools. METHODS: Stool specimens received from nine US laboratories were cultured using media selective for C. difficile, Staphylococcus aureus, vancomycin-resistant enterococci (VRE), and carbapenem-resistant Gram-negative organisms (CROs). Specimens and isolates were also tested by polymerase chain reaction (PCR). Bacterial isolates underwent susceptibility testing and genotyping. FINDINGS: Among 363 specimens, 175 yielded toxigenic C. difficile isolates spanning 27 PCR ribotypes. C. difficile (TCD+) stools harboured an additional 28 organisms, including six CROs (3.4%), of which two (1.1%) were carbapenemase-producing organisms (CPOs), 19 VRE (10.9%), and three meticillin-resistant S. aureus isolates (MRSA, 1.7 %). Stools that were culture negative for toxigenic C. difficile (TCD-) yielded 26 organisms, including four CROs (2.1%), 20 VRE (10.6), and two MRSA (1.1%). Excluding C. difficile, no significant differences were seen in the rates of the MDROs between TCD+ and TCD- specimens. CONCLUSION: Overall, 15.4% of the TCD+ stools and 11.2% of the TCD- stools carried at least one non-C. difficile MDRO pathogen, indicating that multiple MDROs may be present in the gastrointestinal tracts of patients, including those that harbour C. difficile.

Whole genome sequencing options for bacterial strain typing and epidemiologic analysis based on single nucleotide polymorphism versus gene-by-gene-based approaches.

BACKGROUND: Whole genome sequence (WGS)-based strain typing finds increasing use in the epidemiologic analysis of bacterial pathogens in both public health as well as more localized infection control settings. AIMS: This minireview describes methodologic approaches that have been explored for WGS-based epidemiologic analysis and considers the challenges and pitfalls of data interpretation. SOURCES: Personal collection of relevant publications. CONTENT: When applying WGS to study the molecular epidemiology of bacterial pathogens, genomic variability between strains is translated into measures of distance by determining single nucleotide polymorphisms in core genome alignments or by indexing allelic variation in hundreds to thousands of core genes, assigning types to unique allelic profiles. Interpreting isolate relatedness from these distances is highly organism specific, and attempts to establish species-specific cutoffs are unlikely to be generally applicable. In cases where single nucleotide polymorphism or core gene typing do not provide the resolution necessary for accurate assessment of the epidemiology of bacterial pathogens, inclusion of accessory gene or plasmid sequences may provide the additional required discrimination. IMPLICATIONS: As with all epidemiologic analysis, realizing the full potential of the revolutionary advances in WGS-based approaches requires understanding and dealing with issues related to the fundamental steps of data generation and interpretation.

Wide geographical dissemination of the multiresistant Staphylococcus capitis NRCS-A clone in neonatal intensive-care units.

Nosocomial late-onset sepsis represents a frequent cause of morbidity and mortality in preterm neonates. The Staphylococcus capitis clone NRCS-A has been previously described as an emerging cause of nosocomial bacteraemia in French neonatal intensive-care units (NICUs). In this study, we aimed to explore the possible unrecognized dissemination of this clone on a larger geographical scale. One hundred methicillin-resistant S. capitis strains isolated from neonates (n = 86) and adult patients (n = 14) between 2000 and 2013 in four different countries (France, Belgium, the UK, and Australia) were analysed with SmaI pulsed-field gel electrophoresis (PFGE) and dru typing. The vast majority of NICU strains showed the NRCS-A pulsotype and the dt11c type (96%). We then randomly selected 14 isolates (from neonates, n = 12, three per country; from adult patients, n = 2), considered to be a subset of representative isolates, and performed further molecular typing (SacII PFGE, SCCmec typing, and multilocus sequence typing-like analysis), confirming the clonality of the S. capitis strains isolated from neonates, despite their distant geographical origin. Whole genome single-nucleotide polymorphism-based phylogenetic analysis of five NICU isolates (from the different countries) attested to high genetic relatedness within the NRCS-A clone. Finally, all of the NRCS-A strains showed multidrug resistance (e.g. methicillin and aminoglycoside resistance, and decreased vancomycin susceptibility), with potential therapeutic implications for infected neonates. In conclusion, this study represents the first report of clonal dissemination of methicillin-resistant coagulase-negative Staphylococcus clone on a large geographical scale. Questions remain regarding the origin and means of international spread, and the reasons for this clone's apparent predilection for neonates.

ST2249-MRSA-III: a second major recombinant methicillin-resistant Staphylococcus aureus clone causing healthcare infection in the 1970s.

Typing of healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) from Australia in the 1970s revealed a novel clone, ST2249-MRSA-III (CC45), present from 1973 to 1979. This clone was present before the Australian epidemic caused by the recombinant clone, ST239-MRSA-III. This study aimed to characterize the genome of ST2249-MRSA-III to establish its relationship to other MRSA clones. DNA microarray analysis was conducted and a draft genome sequence of ST2249 was obtained. The recombinant structure of the ST2249 genome was revealed by comparisons to publicly available ST239 and ST45 genomes. Microarray analysis of genomic DNA of 13 ST2249 isolates showed gross similarities with the ST239 chromosome in a segment around the origin of replication and with ST45 for the remainder of the chromosome. Recombination breakpoints were precisely determined by the changing pattern of nucleotide polymorphisms in the genome sequence of ST2249 isolate SK1585 compared with ST239 and ST45. One breakpoint was identified to the right of oriC, between sites 1014 and 1065 of the gene D484_00045. Another was identified to the left of oriC, between sites 1185 and 1248 of D484_01632. These results indicate that ST2249 inherited approximately 35.3% of its chromosome from an ST239-like parent and 64.7% from an ST45-like parent. ST2249-MRSA-III resulted from a major recombination between parents that resemble ST239 and ST45. Although only limited Australian archival material is available, the oldest extant isolate of ST2249 predates the oldest Australian isolate of ST239 by 3 years. It is therefore plausible that these two recombinant clones were introduced into Australia separately.

Molecular characterization of methicillin-resistant Staphylococcus aureus isolated over a 2-year period in a Qatari hospital from multinational patients.

Global spread of epidemic methicillin-resistant Staphylococcus aureus (MRSA) is an issue of increasing clinical concern especially problematic community-associated (CA) -MRSA. However, data regarding MRSA epidemiology in regions of the Middle East, including Qatar, are still insufficient. A representative subset of 61 MRSA isolates from multinational patients from hospital in Qatar during a 2-year period (2009/2010) was examined. Molecular characterization for MRSA isolates was performed by pulsed-field gel electrophoresis (PFGE), SCCmec, spa and dru typing, and PCR for the presence of the arginine catabolic mobile element and genes for the Panton-Valentine leukocidin (PVL). Prevalence of MRSA among S. aureus isolated was 176/840 (21%). Of the 61 MRSA isolates examined, three (5%) represented hospital-acquired infection. By PFGE, 32 isolates (52%) were CA-MRSA USA300 (n = 4), USA400 (n = 3), USA1100/Southwest (SW) Pacific (n = 17) and ST80-MRSA-IV (n = 8) strains. The remaining isolates were well-known healthcare-associated EMRSA-15 (n = 8) and USA800 (n = 13). Three isolates were USA900, one was USA1200 and four were unrelated to any known strains in the international database. Unexpectedly, the USA900 isolates were all positive for PVL and USA400 isolates were PVL negative. Five of the eight EMRSA-15 isolates were PVL positive. ST80-MRSA-IV and USA300 strains exhibited typical dru types (dt10a and dt9g, respectively). Eleven different spa types were observed in this study. All USA300 isolates were arginine catabolic mobile element positive. The high prevalence of CA-MRSA, especially including USA300, in this setting underscores the importance of global epidemiological monitoring to better understand and hopefully help prevent the emergence and spread of these problem pathogens in patient populations.

Characterization of a novel composite staphylococcal cassette chromosome mec (SCCmec-SCCcad/ars/cop) in the neonatal sepsis-associated Staphylococcus capitis pulsotype NRCS-A.

Multiresistant Staphylococcus capitis pulsotype NRCS-A has been reported to be a major pathogen causing nosocomial bacteremia in preterm infants. We report that the NRCS-A strain CR01 harbors a novel 60.9-kb composite staphylococcal cassette chromosome mec (SCCmec) element, composed of an SCCmec with strong homologies to Staphylococcus aureus ST398 SCCmec and of an SCCcad/ars/cop harboring resistance genes for cadmium, arsenic, and copper. Whole-genome-based comparisons of published S. capitis strains suggest that strain CR01 acquired the two elements independently.

Daptomycin non-susceptible Staphylococcus aureus at a US medical centre.

Daptomycin non-susceptible Staphylococcus aureus was rarely encountered at our medical centre until 2010, when 10 isolates (0.4% of S. aureus) were confirmed by E-test as non-susceptible. These isolates were not of the same strain type and there was no link between the 10 patients. Daptomycin non-susceptibility may be increasing.

From theory to practice: molecular strain typing for the clinical and public health setting.

The persistence and transmission of infectious disease is one of the most enduring and daunting concerns in healthcare. Over the years, epidemiological analysis especially of bacterial etiological agents has undergone a remarkable evolutionary metamorphosis. While initially relying on purely phenotypic characterisation, advances in molecular biology have found translational application in a number of approaches to strain typing which commonly centre either on 'epityping' (molecular epidemiology) to characterise outbreaks, perform surveillance, and trace evolutionary pathways, or 'pathotyping' to compare strains based on the presence or absence of specific virulence or resistance genes. A perspective overview of strain typing is presented here considering the issues surrounding analyses which are employed in the localised clinical setting as well as at a more regional/national public health level. The discussion especially considers the shortcomings inherent in epidemiological analysis: less than full isolate characterisation by the typing method and limitations imposed by the available data, context, and time constraints of the epidemiological investigation (i.e. the available epidemiological window). However, the promises outweigh the pitfalls as one considers the potential for advances in genomic characterisation and information technology to provide an unprecedented aggregate of epidemiological information and analysis.

Methicillin sensitive Staphylococcus aureus (MSSA) isolates as a potential source for the emergence of USA 300 methicillin resistant Staphylococcus aureus (MRSA) in Malaysia.

We investigated the potential of USA300 MRSA emergence in Malaysia by examining 268 MSSA isolates from both community (110) and healthcare (158) settings. Nine isolates from both the environments were similar to the USA300 MRSA background based on MLST, spa and PFGE type. These results underscore the importance of continued surveillance to monitor the emergence of USA300 MRSA in Malaysia.

Two novel class I integron arrays containing IMP-18 metallo-ß-lactamase gene in Pseudomonas aeruginosa clinical isolates from Puerto Rico.

During a ß-lactam resistance surveillance study, 12 IMP-18-positive Pseudomonas aeruginosa isolates belonging to 9 different pulsed-field gel electrophoresis groups were identified. In nine isolates, a class I integron with a novel gene array was identified that contained bla(IMP-18) and bla(OXA-224), while in two isolates the class I integron contained bla(IMP-18) and bla(OXA-2) but in a new arrangement. Our findings show the dissemination of two novel class I integrons in P. aeruginosa from different regions of Puerto Rico.

mec-associated dru typing in the epidemiological analysis of ST239 MRSA in Malaysia.

The usefulness of mec-associated dru typing in the epidemiological analysis of methicillin-resistant Staphylococcus aureus (MRSA) isolated in Malaysia was investigated and compared with pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and spa and SCCmec typing. The isolates studied included all MRSA types in Malaysia. Multilocus sequence type ST188 and ST1 isolates were highly clonal by all typing methods. However, the dru typing of ST239 isolates produced the clearest discrimination between SCCmec IIIa and III isolates, yielding more subtypes than any other method. Evaluation of the discriminatory power for each method identified dru typing and PFGE as the most discriminatory, with Simpson's index of diversity (SID) values over 89%, including an isolate which was non-typeable by spa, but dru-typed as dt13j. The discriminatory ability of dru typing, especially with closely related MRSA ST239 strains (e.g., Brazilian and Hungarian), underscores its utility as a tool for the epidemiological investigation of MRSA.

Comparative genomic analysis of European and Middle Eastern community-associated methicillin-resistant Staphylococcus aureus (CC80:ST80-IV) isolates by high-density microarray.

Infections as a result of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are an issue of increasing global healthcare concern. In Europe, this principally involves strains of multi-locus sequence type clonal complex 80 sequence type 80 with methicillin resistance in a staphylococcal chromosomal cassette (SCCmec) type IV arrangement (CC80:ST80-IV). As with other CA-MRSA strains, CC80:ST80-IV isolates tend to appear uniform when analysed by common molecular typing methods (e.g. pulsed field gel electrophoresis, multi-locus sequence type, SCCmec). To explore whether DNA sequence-based differences exist, we compared the genetic composition of six CC80:ST80-IV isolates of diverse chronological and geographic origin (i.e. Denmark and the Middle East) using an Affymetrix high-density microarray that was previously used to analyse CA-MRSA USA300 isolates. The results revealed a high degree of homology despite the diversity in isolation date and origin, with isolate differences primarily in conserved hypothetical open reading frames and intergenic sequences, but also including regions of known function. This included the confirmed loss of SCCmec recombinase genes in two Danish isolates representing potentially new SCCmec types. Microarray analysis grouped the six isolates into three relatedness pairs, also identified by pulsed field gel electrophoresis, which were consistent with both the clinical and molecular data.

Usefulness of mec-associated direct repeat unit (dru) typing in the epidemiological analysis of highly clonal methicillin-resistant Staphylococcus aureus in Scotland.

The incidence of the epidemic methicillin-resistant Staphylococcus aureus (EMRSA) strains EMRSA-15 and EMRSA-16 in Scotland has increased dramatically, now accounting for c. 70% and c. 20% of isolates, respectively. Epidemiological tracking of these EMRSA strains is difficult, as c. 50% of EMRSA-15 and c. 35% of EMRSA-16 isolates are indistinguishable using pulsed-field gel electrophoresis (PFGE) and other typing methods. The usefulness of mec-associated direct repeat unit (dru) sequence analysis as a more sensitive approach to tracking the persistence and spread of these 'clonal' EMRSA strains in Scotland was evaluated. Analysis of 47 EMRSA-15 and 57 EMRSA-16 isolates (including two separately cultured isolates of the Harmony collection type strain) obtained from 22 hospital laboratories over an 8-year period (1997-2005) revealed 13 and 12 different dru types, respectively. Whereas some types appeared to be endemic in multiple hospitals, subtypes that may represent specific strain movement among hospitals in a given geographical region were identified in other instances. These results suggest that mec-associated dru typing may have potential for identifying and tracking specific subtypes of otherwise indistinguishable epidemic MRSA isolates such as those in Scotland.

Epidemiology of European community-associated methicillin-resistant Staphylococcus aureus clonal complex 80 type IV strains isolated in Denmark from 1993 to 2004.

In Europe, community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections have been caused predominantly by isolates belonging to the "European CA-MRSA" clone (sequence type 80, staphylococcal cassette chromosome mec type IV). In this study, the epidemiology of European CA-MRSA was investigated on a nationwide scale, covering the period from 1993 to 2004. Denmark has been a low-prevalence country regarding MRSA since the mid-1970s but has experienced an increase in the number of new MRSA cases in recent years. Our results show that European CA-MRSA contributed to this increase. The isolates primarily caused skin and soft tissue infections (SSTIs) in patients outside hospitals, and transmission between household members was the predominant mode of spread. Although some of the isolates were found in hospitalized patients, nosocomial transmission seemed likely in only one instance, pointing to endogenous infections as an important factor. Compared to the CA-MRSA clone most common in the United States (USA300), the European CA-MRSA clone seems less well adapted to persist in hospital environments. Patients with a recent history of travel or family relation to the Mediterranean or Middle East were highly overrepresented. The epidemiological data indicated that the European CA-MRSA isolates were introduced into Denmark on multiple occasions, paralleled by an increasing level of genetic diversity of the isolates found during the study period. European CA-MRSA has previously been described as a rather uniform clone. However, we found pronounced, diverse pulsed-field gel electrophoresis subtypes, staphylococcal protein A gene (spa) types, and susceptibility patterns.

Allelic variation in genes encoding Panton-Valentine leukocidin from community-associated Staphylococcus aureus.

Community-associated methicillin-resistant Staphylococcus aureus isolates characteristically contain the genes for Panton-Valentine leukocidin (PVL), which is a proposed virulence factor. To determine whether different alleles of the PVL genes lukS-PV and lukF-PV occur, and whether they are associated with specific genetic lineages of S. aureus, sequences from 28 S. aureus isolates, representing four different multilocus sequence types, and bacteriophages SLT and PVL were compared. Seven nucleotide polymorphisms were identified, which defined three groups of the lukS-PV and lukF-PV sequence. Only one polymorphism resulted in an amino-acid change. Bacteriophage SLT and isolates of bacteriophage type 80/81 contained the prototypic (founder) lukS-PV and lukF-PV sequence. The alleles were not lineage-specific.

Plasmid-mediated carbapenem-hydrolyzing enzyme KPC-2 in an Enterobacter sp.

A strain of an Enterobacter sp. with reduced susceptibility to imipenem, which produced a plasmid-mediated class A carbapenem-hydrolyzing enzyme, KPC-2 beta-lactamase, was isolated from a patient with sepsis at a Boston hospital. This is the first report of the production of a plasmid-encoded KPC-2 beta-lactamase by an Enterobacter sp.

EmtA, a rRNA methyltransferase conferring high-level evernimicin resistance.

Enterococcus faecium strain 9631355 was isolated from animal sources on the basis of its resistance to the growth promotant avilamycin. The strain also exhibited high-level resistance to evernimicin, a drug undergoing evaluation as a therapeutic agent in humans. Ribosomes from strain 9631355 exhibited a dramatic reduction in evernimicin binding, shown by both cell-free translation assays and direct-binding assays. The resistance determinant was cloned from strain 9631355; sequence alignments suggested it was a methyltransferase and therefore it was designated emtA for evernimicin methyltransferase. Evernimicin resistance was transmissible and emtA was localized to a plasmid-borne insertion element. Purified EmtA methylated 50S subunits from an evernimicin-sensitive strain 30-fold more efficiently than those from a resistant strain. Reverse transcription identified a pause site that was unique to the 23S rRNA extracted from resistant ribosomes. The pause corresponded to methylation of residue G2470 (Escherichia coli numbering). RNA footprinting revealed that G2470 is located within the evernimicin-binding site on the ribosome, thus providing an explanation for the reduced binding of the drug to methylated ribosomes.

Molecular typing of methicillin-resistant Staphylococcus aureus by pulsed-field gel electrophoresis: comparison of results obtained in a multilaboratory effort using identical protocols and MRSA strains.

Pulsed-field gel electrophoresis (PFGE) has become the gold standard of molecular methods in epidemiological investigations. In spite of its high resolving power, use of the method has been hampered by inadequate laboratory-to-laboratory reproducibility. In the project described here we have addressed this problem by organizing a multilaboratory effort in which the same bacterial strains (subtype variants of the Iberian and Brazilian methicillin-resistant Staphylococcus aureus--MRSA--clones) were analyzed by twenty investigators in thirteen different laboratories according to an indentical protocol, which is reproduced here in detail. PFGE patterns obtained were analyzed at a central laboratory in order to identify specific technical problems that produced substandard macrorestriction patterns. The results including the specific technical problems and their most likely causes are described in this communication. Also listed are seven major epidemic clones of MRSA which have been characterized by molecular fingerprinting techniques and the prototypes of which have been deposited at the American Type Culture Collection, from where they will be available for interested investigators for the purpose of typing MRSA isolates. It is hoped that this communication will contribute to the improvement of the reproducibility and technical/aesthetic quality of PFGE analysis.

In vitro activity of evernimicin and selected antibiotics against methicillin-resistant staphylococci: a 24-country study.

OBJECTIVE: To collect and analyze data on susceptibility of methicillin-resistant staphylococci to evernimicin and other antimicrobial agents. METHODS: Recent clinical isolates of methicillin-resistant staphylococci from 33 laboratories in North America, Europe and South Africa were investigated. RESULTS: Of the antimicrobial agents tested, evernimicin had the lowest MIC90s for methicillin-resistant Staphylococcus aureus and methicillin-resistant coagulase-negative staphylococci (0.75 and 1.0 mg/L, respectively). Resistance to ciprofloxacin and erythromycin was widespread, with higher levels of resistance in North America than in other regions. CONCLUSIONS: Susceptibility surveys help to determine the antimicrobial activity of new agents. Ciprofloxacin- and erythromycin-resistant staphylococci were prevalent throughout all regions.