Characterization of new staphylococcal cassette chromosome mec (SCCmec) and topoisomerase genes in fluoroquinolone- and methicillin-resistant Staphylococcus pseudintermedius

Fluoroquinolone- and methicillin-resistant Staphylococcus pseudintermedius isolates harbor two new staphylococcal cassette chromosome mec (SCCmec) elements that belong to class A, allotype 3 (SCCmec II-III), and to the new allotype 5 (SCCmec VII). Analysis of the complete nucleotide sequences of the topoisomerase loci gyrB/gyrA and grlB/grlA revealed mutations involved in fluoroquinolone resistance.

Mutations upstream of fabI in triclosan resistant Staphylococcus aureus strains are associated with elevated fabI gene expression

Background The enoyl-acyl carrier protein (ACP) reductase enzyme (FabI) is the target for a series of antimicrobial agents including novel compounds in clinical trial and the biocide triclosan. Mutations in fabI and heterodiploidy for fabI have been shown to confer resistance in S. aureus strains in a previous study. Here we further determined the fabI upstream sequence of a selection of these strains and the gene expression levels in strains with promoter region mutations. Results Mutations in the fabI promoter were found in 18% of triclosan resistant clinical isolates, regardless the previously identified molecular mechanism conferring resistance. Although not significant, a higher rate of promoter mutations were found in strains without previously described mechanisms of resistance. Some of the mutations identified in the clinical isolates were also detected in a series of laboratory mutants. Microarray analysis of selected laboratory mutants with fabI promoter region mutations, grown in the absence of triclosan, revealed increased fabI expression in three out of four tested strains. In two of these strains, only few genes other than fabI were upregulated. Consistently with these data, whole genome sequencing of in vitro selected mutants identified only few mutations except the upstream and coding regions of fabI, with the promoter mutation as the most probable cause of fabI overexpression. Importantly the gene expression profiling of clinical isolates containing similar mutations in the fabI promoter also showed, when compared to unrelated non-mutated isolates, a significant up-regulation of fabI. Conclusions In conclusion, we have demonstrated the presence of C34T, T109G, and A101C mutations in the fabI promoter region of strains with fabI up-regulation, both in clinical isolates and/or laboratory mutants. These data provide further observations linking mutations upstream fabI with up-regulated expression of the fabI gene.

Effectiveness of meticillin-resistant Staphylococcus aureus decolonization in long-term haemodialysis patients: a systematic review and meta-analysis.

BACKGROUND Chronic haemodialysis patients are a high-risk population for meticillin-resistant Staphylococcus aureus (MRSA) colonization, which is a precursor of infection. AIM To summarize the effect of nasal (± whole-body wash) MRSA decolonization in haemodialysis patients by means of a systematic review and meta-analysis. METHODS We identified eligible studies using Medline, Embase, the Cochrane database, clinicaltrials.org, and conference abstracts investigating the success of MRSA decolonization in haemodialysis patients. For the statistical analysis, we used Stata 13 to express study-specific proportions with 95% confidence intervals. A likelihood ratio test was used to assess inter-study heterogeneity. FINDINGS Six published prospective cohort studies and one study described in a conference abstract met our inclusion criteria. From 1150 haemodialysis patients enrolled in these studies, MRSA was isolated from nasal swabs of 147 (12.8%) patients. Six of the trials used mupirocin nasal ointment and combined it with chlorhexidine body washes for decolonization. The most widely used protocol was a five-day course of mupirocin nasal ointment application three times a day, and chlorhexidine body wash once daily. The pooled success rate of decolonization was 0.88 (95% confidence interval: 0.75-0.95). A likelihood ratio test of the fixed versus the random-effects model showed significant inter-study heterogeneity (P = 0.047). Four of seven studies determined subsequent MRSA infections in 94 carriers overall, two (2%) of which experienced infection. CONCLUSION The use of mupirocin together with whole-body decolonization is highly effective in eradicating MRSA carriage in haemodialysis patients. The current literature, however, is characterized by a lack of comparative effectiveness studies for this intervention.

Characterization of a Novel Composite Staphylococcal Cassette Chromosome mec in Methicillin-Resistant Staphylococcus pseudintermedius from Thailand.

A novel staphylococcal cassette chromosome mec (SCCmec) composite island (SCCmecAI16-SCCczrAI16-CI) was identified in Staphylococcus pseudintermedius. Four integration site sequences for SCC subdivided the 60,734-bp island into 41,232-bp SCCmecAI16, 19,400-bp SCCczrAI16, and 102-bp SCC-likeAI16 elements. SCCmecAI16 represents a new combination of ccrA1B3 genes with a class A mec complex. SCCczrAI16 contains ccrA1B6 and genes related to restriction modification and heavy metal resistance. SCCmecAI16-SCCczrAI16-CI was found in methicillin-resistant S. pseudintermedius sequence type 112 (ST112) and ST111 isolated from dogs and veterinarians in Thailand.

Microarray based study on virulence-associated genes and resistance determinants of Staphylococcus aureus isolates from cattle

Staphylococcus aureus is a common pathogen which can colonise and infect not only man, but also domestic animals. Especially, infection of cattle is of high economic relevance as S. aureus is an important causal agent of bovine mastitis. In the present contribution, a DNA microarray was applied for the study of 144 different gene targets, including resistance genes and genes encoding exotoxins, in S. aureus isolated from cows. One hundred and twenty-eight isolates from Germany and Switzerland were tested. These isolates were assigned to 20 different strains and nine clonal complexes. The majority of isolates belonged either to apparently closely related clonal complexes 8, 25, and 97 (together 34.4%) or were related to the sequenced bovine strain RF122 (48.4%). Notable characteristics of S. aureus of bovine origin are the carriage of intact haemolysin beta (in 82% of isolates tested), the absence of staphylokinase (in 89.1%), the presence of allelic variants of several exotoxins such as toxic shock syndrome toxin and enterotoxin N, and the occurrence of the leukocidin lukF-P83/lukM (in 53.1%). Two isolates were methicillin-resistant S. aureus (MRSA). One of them was a clonal complex 8 MRSA related to the epidemic MRSA strain Irish 01. The other one belonged to ST398/spa-type 34 resembling a newly emerging MRSA strain which has been described to occur in humans as well as in domestic animals. The presence of these two strains highlights the possibility of transfers of S. aureus strains between different host species.

Genetic diversity and ecological success of Staphylococcus aureus strains colonizing humans

The genetic determinants and phenotypic traits which make a Staphylococcus aureus strain a successful colonizer are largely unknown. The genetic diversity and population structure of 133 S. aureus isolates from healthy, generally risk-free adult carriers were investigated using four different typing methods: multilocus sequence typing (MLST), amplified fragment length polymorphism analysis (AFLP), double-locus sequence typing (DLST), and spa typing were compared. Carriage isolates displayed great genetic diversity which could only be revealed fully by DLST. Results of AFLP and MLST were highly concordant in the delineation of genotypic clusters of closely related isolates, roughly equivalent to clonal complexes. spa typing and DLST provided considerably less phylogenetic information. The resolution of spa typing was similar to that of AFLP and inferior to that of DLST. AFLP proved to be the most universal method, combining a phylogeny-building capacity similar to that of MLST with a much higher resolution. However, it had a lower reproducibility than sequencing-based MLST, DLST, and spa typing. We found two cases of methicillin-resistant S. aureus colonization, both of which were most likely associated with employment at a health service. Of 21 genotypic clusters detected, 2 were most prevalent: cluster 45 and cluster 30 each colonized 24% of the carrier population. The number of bacteria found in nasal samples varied significantly among the clusters, but the most prevalent clusters were not particularly numerous in the nasal samples. We did not find much evidence that genotypic clusters were associated with different carrier characteristics, such as age, sex, medical conditions, or antibiotic use. This may provide empirical support for the idea that genetic clusters in bacteria are maintained in the absence of adaptation to different niches. Alternatively, carrier characteristics other than those evaluated here or factors other than human hosts may exert selective pressure maintaining genotypic clusters.

Strain typing and antimicrobial susceptibility of methicillin-resistant coagulase-positive staphylococcal species in dogs and people associated with dogs in Thailand

AIMS This study was to investigate and to characterize methicillin-resistant coagulase-positive staphylococci (MRCoPS) harboring in dogs and people associated with dogs in Thailand. METHODS AND RESULTS Staphylococci were collected from 100 dogs, 100 dog owners, 200 small animal veterinarians and 100 people without pet association. Species of MRCoPS were identified phenotypically and genotypically. Molecular characteristics were determined by multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE) and SCCmec typing, and antimicrobial susceptibility was assayed by broth microdilution and by microarray analysis for resistance genes. Methicillin-resistant Staphylococcus pseudintermedius (MRSP), methicillin-resistant Staphylococcus schleiferi subsp. coagulans (MRSSc) and methicillin-resistant Staphylococcus aureus (MRSA) were isolated from dogs (45, 17 and 1%, respectively), veterinarians (8, 2 and 1·5%, respectively) and dog owners (3, 2 and 0%, respectively). Seventeen sequence types (STs) were identified among 83 MRSP isolates which specifically carried SCCmec V, II-III, ΨSCCmec57395 and three uncharacterized SCCmec types. MRSP ST 45, 68 and novel STs including 169, 178, 181 and 183 were shared among canine and human isolates. Most of MRSA ST398 and MRSSc carried SCCmec type V. The MRCoPS commonly displayed multiple resistances to tested antimicrobials and carried various resistance genes. CONCLUSION Variety of MRCoPS, especially new MRSP clones, distributed in dogs and people in Thailand. SIGNIFICANCE AND IMPACT OF THE STUDY The existence of MRCoPS circulating between dogs and humans in Thailand provides indirect evidence of interspecies transmission and represents a potential public health hazard.

Colonization with resistant microorganisms in patients transferred from abroad: who needs to be screened?

BACKGROUND While multi-drug resistant organisms (MDRO) are a global phenomenon, there are significant regional differences in terms of prevalence. Traveling to countries with a high MDRO prevalence increases the risk of acquiring such an organism. In this study we determined risk factors for MDRO colonization among patients who returned from a healthcare system in a high-prevalence area (so-called transfer patients). Factors predicting colonization could serve as screening criteria to better target those at highest risk. METHODS This screening study included adult patients who had been exposed to a healthcare system abroad or in a high-prevalence region in Switzerland over the past six months and presented to our 950-bed tertiary care hospital between January 1, 2012 and December 31, 2013, a 24-month period. Laboratory screening tests focused on Gram-negative MDROs and methicillin-resistant Staphylococcus aureus (MRSA). RESULTS A total of 235 transfer patients were screened and analyzed, of which 43 (18 %) were positive for an MDRO. Most of them yielded Gram-negative bacteria (42; 98 %), with only a single screening revealing MRSA (2 %); three screenings showed a combination of Gram-negative bacteria and MRSA. For the risk factor analysis we focused on the 42 Gram-negative MDROs. Most of them were ESBL-producing Escherichia coli and Klebsiella pneumoniae while only two were carbapenemase producers. In univariate analysis, factors associated with screening positivity were hospitalization outside of Europe (p < 0.001), surgical procedure in a hospital abroad (p = 0.007), and - on admission to our hospital - active infection (p = 0.002), antibiotic treatment (p = 0.014) and presence of skin lesions (p = 0.001). Only hospitalization outside of Europe (Odds Ratio, OR 3.2 (95 % CI 1.5- 6.8)) and active infection on admission (OR 2.7 (95 % CI 1.07- 6.6)) remained as independent predictors of Gram-negative MDRO colonization. CONCLUSION Our data suggest that a large proportion of patients (i.e., 82 %) transferred to Switzerland from hospitals in high MDRO prevalence areas are unnecessarily screened for MDRO colonization. Basing our screening strategy on certain criteria (such as presence of skin lesions, active infection, antibiotic treatment, history of a surgical procedure abroad and hospitalization outside of Europe) promises to be a better targeted and more cost-effective strategy.

Variability of SCCmec in the Zurich area

A periodic survey of methicillin-resistant Staphylococcus aureus (MRSA) in Zurich in 2004 and 2006 revealed a consistently low prevalence of MRSA. SCCmec and ccr typing showed fluctuations in the proportions of SCCmec types and in the carriage of mobile virulence determinants. Together with the presence of variant SCCmecs these findings suggest a high clonal diversity and level of SCCmec recombination. The prevalence of a local "drug clone", associated with low-level methicillin resistance and rapid growth, significantly decreased. This clone had spread among intraveneous drug users, steadily increasing from 1994 to 2001 and was dominant in 2001. Apparently, changes in the management of the Zurich drug scene have restricted the spread of this clone.

Pressure Ulcer-Related Pelvic Osteomyelitis: A Neglected Disease?

Background. Decubitus ulcers can become complicated by pelvic osteomyelitis. Little is known about the epidemiology of pressure ulcer-related pelvic osteomyelitis. Methods. We performed a retrospective cohort study of adult patients with pressure ulcer and pelvic osteomyelitis admitted to an academic center from 2006 to 2011. Data on clinical presentation, diagnostic evaluation, and treatment during the index admission were collected. Outcome measures included length of hospital stay and number of readmissions in the subsequent year. Results. Two hundred twenty patients were included: 163 (74%) were para/quadriplegic and 148 (67%) were male (148; 67%). Mean age was 50 (±18) years. Pelvic osteomyelitis was the primary admission diagnosis for 117 (53%). Fifty-six (26%) patients had concurrent febrile urinary tract infection. Wound cultures collected for 113 patients (51%) were notable for methicillin-resistant Staphylococcus aureus (37; 33%), Streptococci (19; 17%), and Pseudomonas spp (20; 18%). Plain films were obtained in 89 (40%) patients, computed tomography scans were obtained for 81 (37%) patients, and magnetic resonance images were obtained for 40 (18%) patients. Most patients received osteomyelitis-directed antibiotics (153; 70%), 134 of 153 (88%) of which were scheduled to receive ≥6 weeks of treatment. Fifty-five (25%) patients underwent surgery during the index admission; 48 (22%) patients received a combined medical-surgical approach. One third of patients had ≥2 readmissions during the subsequent year. Patients treated with a combined approach were less likely to be readmitted than those who received antibiotics alone (0 [range, 0-4] vs 1 [0-7] readmissions; P = .04). Conclusions. This is one of the largest cohort studies of pressure ulcer-related pelvic osteomyelitis to date. Significant variations existed in diagnostic approach. Most patients received antibiotics; those treated with a combined medical-surgical approach had fewer hospital readmissions.

Efficacy of telavancin against penicillin-resistant pneumococci and Staphylococcus aureus in a rabbit meningitis model and determination of kinetic parameters

The penetration of telavancin was 2% into inflamed meninges and ca. 1 per thousand into noninflamed meninges after two intravenous injections (30 mg/kg of body weight). In experimental meningitis, telavancin was significantly superior to vancomycin combined with ceftriaxone against a penicillin-resistant pneumococcal strain. Against a methicillin-sensitive staphylococcal strain, telavancin was slightly but not significantly superior to vancomycin.

Differentiation between Staphylococcus aureus and coagulase-negative Staphylococcus species by real-time PCR including detection of methicillin resistants in comparison to conventional microbiology testing.

BACKGROUND Staphylococcus aureus has long been recognized as a major pathogen. Methicillin-resistant strains of S. aureus (MRSA) and methicillin-resistant strains of S. epidermidis (MRSE) are among the most prevalent multiresistant pathogens worldwide, frequently causing nosocomial and community-acquired infections. METHODS In the present pilot study, we tested a polymerase chain reaction (PCR) method to quickly differentiate Staphylococci and identify the mecA gene in a clinical setting. RESULTS Compared to the conventional microbiology testing the real-time PCR assay had a higher detection rate for both S. aureus and coagulase-negative Staphylococci (CoNS; 55 vs. 32 for S. aureus and 63 vs. 24 for CoNS). Hands-on time preparing DNA, carrying out the PCR, and evaluating results was less than 5 h. CONCLUSIONS The assay is largely automated, easy to adapt, and has been shown to be rapid and reliable. Fast detection and differentiation of S. aureus, CoNS, and the mecA gene by means of this real-time PCR protocol may help expedite therapeutic decision-making and enable earlier adequate antibiotic treatment.

Genetic Relatedness, Antimicrobial and Biocide Susceptibility Comparative Analysis of Methicillin-Resistant and -Susceptible Staphylococcus pseudintermedius from Portugal

Forty methicillin-resistant and -susceptible Staphylococcus pseudintermedius (MRSP and MSSP, respectively) from colonization and infection in dogs and cats were characterized for clonality, antimicrobial, and biocide susceptibility. MSSP were genetically more diverse than MRSP by multi-locus sequence typing and pulsed-field gel electrophoresis. Three different spa types (t06, t02, t05) and two SCCmec types (II-III and V) were detected in the MRSP isolates. All MRSP and two MSSP strains were multidrug-resistant. Several antibiotic resistance genes (mecA, blaZ, tet(M), tet(K), aac(6')-Ie-aph(2')-Ia, aph(3')-III, ant(6)-Ia, sat4, erm(B), lnu(A), dfr(G), and catpC221) were identified by microarray and double mutations in the gyrA and grlA genes and a single mutation in the rpoB gene were detected by sequence analysis. No differences were detected between MSSP and MRSP in the chlorhexidine acetate (CHA) minimum inhibitory concentrations (MICs). However, two MSSP had elevated MIC to triclosan (TCL) and one to benzalkonium chloride and ethidium bromide. One MSSP isolate harboured a qacA gene, while in another a qacB gene was detected. None of the isolates harboured the sh-fabI gene. Three of the biocide products studied had high bactericidal activity (Otodine(®), Clorexyderm Spot Gel(®), Dermocanis Piocure-M(®)), while Skingel(®) failed to achieve a five log reduction in the bacterial counting. S. pseudintermedius have become a serious therapeutic challenge in particular if methicillin- resistance and/or multidrug-resistance are involved. Biocides, like CHA and TCL, seem to be clinically effective and safe topical therapeutic options.

Clinical and molecular features of methicillin-resistant, coagulase-negative staphylococci of pets and horses

OBJECTIVES To determine the antibiotic resistance and fingerprint profiles of methicillin-resistant coagulase-negative staphylococci (MRCoNS) from animal infections among different practices and examine the history of antibiotic treatment. METHODS Isolates were identified by mass spectrometry and tested for antimicrobial resistance by broth dilution, microarrays and sequence analysis of the topoisomerases. Diversity was assessed by PFGE, icaA PCR and staphylococcal cassette chromosome mec (SCCmec), arginine catabolic mobile element (ACME) and multilocus sequence typing. Clinical records were examined retrospectively. RESULTS MRCoNS were identified as Staphylococcus epidermidis (n=20), Staphylococcus haemolyticus (n=17), Staphylococcus hominis (n=3), Staphylococcus capitis (n=1), Staphylococcus cohnii (n=1) and Staphylococcus warneri (n=1). PFGE identified one clonal lineage in S. hominis isolates and several in S. haemolyticus and S. epidermidis. Fourteen sequence types were identified in S. epidermidis, with sequence type 2 (ST2) and ST5 being predominant. Ten isolates contained SCCmec IV, seven contained SCCmec V and the others were non-typeable. ACMEs were detected in 11 S. epidermidis isolates. One S. hominis and 10 S. epidermidis isolates were icaA positive. In addition to mecA-mediated β-lactam resistance, the most frequent resistance was to gentamicin/kanamycin [aac(6')-Ie-aph(2')-Ia, aph(3')-III] (n=34), macrolides/lincosamides [erm(C), erm(A), msr, lnu(A)] (n=31), tetracycline [tet(K)] (n=22), streptomycin [str, ant(6)-Ia] (n=20), trimethoprim [dfr(A), dfr(G)] (n=17), sulfamethoxazole (n = 34) and fluoroquinolones [amino acid substitutions in GyrA and GrlA] (n=30). Clinical data suggest selection through multiple antibiotic courses and emphasize the importance of accurate diagnosis and antibiograms. CONCLUSIONS MRCoNS from animal infection sites are genetically heterogeneous multidrug-resistant strains that represent a new challenge in the prevention and therapy of infections in veterinary clinics.