What causes decreased erythromycin resistance in Streptococcus pyogenes? Dynamics of four clones in a southern European region from 2005 to 2012.

OBJECTIVES: To survey antibiotic resistance among Streptococcus pyogenes isolates collected from 2005 to 2012, to characterize those showing erythromycin resistance and to analyse the association of certain emm types with erythromycin resistance or susceptibility. METHODS: Resistance determinants or mutations conferring erythromycin, clindamycin, tetracycline and fluoroquinolone resistance were analysed. All erythromycin-resistant isolates and a sample of erythromycin-susceptible isolates were emm typed. Multilocus sequence typing was performed for representative emm types. RESULTS: Antimicrobial susceptibility was studied for 12 346 S. pyogenes isolates. Erythromycin, clindamycin and tetracycline resistance showed a decreasing trend. In 2012, 2.8% of isolates were erythromycin resistant versus 7.5% in 2005 and 11.7% in 2006. Although 21 clones were involved, 4 clones accounted for almost 90% of erythromycin-resistant isolates. The emm12/ST36 clone, carrying the mef(A) gene, was the predominant (41.1%) erythromycin-resistant clone, with an incidence peak in 2008, followed by a gradual decline. The M phenotype predominated each year except for 2005, when two of the main erythromycin-resistant clones (emm11/ST403 and emm28/ST52) harboured an erm(B) gene. Erythromycin resistance was significantly higher in adults than in children. Skin isolates showed the highest erythromycin resistance rate; among these, perianal isolates frequently belonged to the emm28/ST52 clone. The emm type was not a predictor of erythromycin resistance; however, most emm11 and emm12 were erythromycin-resistant isolates. Macrolide consumption was similar throughout the study period. Only two isolates with a high level of levofloxacin resistance were detected. CONCLUSIONS: Resistance was mainly related to the circulation of emm12/ST36, emm11/ST403, emm28/ST52 and emm4/ST39 clones, all of which declined throughout the study period.

Decline and rise of the antimicrobial susceptibility of Streptococcus pneumoniae isolated from middle ear fluid in children: influence of changes in circulating serotypes.

Changes in the antimicrobial susceptibility of Streptococcus pneumoniae causing otitis media were studied in 916 isolates from children <5 years old between 1999 and 2010 in a region of northern Spain. The rate of antimicrobial resistance decreased between the period before the introduction of the heptavalent pneumococcal conjugate vaccine (from 1999 to 2001) and the period from 2005 to 2007. However, in 2008 to 2010, resistance rates increased again due to the spread of serotype 19A, especially the multidrug-resistant ST320 and ST276 clones.

Molecular characterization of Staphylococcus aureus carrying the panton-valentine leucocidin genes in northern Spain.

OBJECTIVES: To study the prevalence of the Panton-Valentine leucocidin (PVL) gene in methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus obtained in Gipuzkoa, northeastern area of the Basque Country, north-central Spain, and perform the molecular characterization of PVL-positive isolates. METHODS: Molecular studies comprised: PVL gene detection by PCR, staphylococcal chromosome cassette mec (SCCmec) typing, spa sequencing, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and detection of the arginine catabolic mobile element (ACME). RESULTS: Between 1978 and 2006, only two (0.3%) of the 686 MRSA isolates studied were positive for the PVL gene. This percentage increased between 2007 and 2009, when the PVL gene was detected in 30 of the 679 MRSA (4.4%) and in nine of the 1227 MSSA (0.7%) isolates. The 41 PVL-positive isolates characterized had eight different sequence types (STs). Twenty-three MRSA PVL-positive isolates were ST8, spa type t008, seven of which were ACME positive, erythromycin-resistant and showed the PFGE pattern (90-100% similarity) of the USA300 clone. ST8 was also the most prevalent ST among the nine MSSA PVL-positive isolates. CONCLUSION: The current epidemiology of PVL-positive MRSA in our region more closely resembles that of the USA rather than that of other European countries, being USA300 or USA300-like isolates the most prevalent ones.

Epidemiological and molecular aspects of rifampicin-resistant Staphylococcus aureus isolated from wounds, blood and respiratory samples.

OBJECTIVES: To study the incidence of rifampicin-resistant Staphylococcus aureus in Gipuzkoa, Northern Spain, and to characterize representative resistant isolates and mutations associated with resistance. METHODS: For rifampicin-resistant isolates, the rpoB gene fragment that includes the most frequent mutations conferring rifampicin resistance in S. aureus was amplified and sequenced. The role of new mutations responsible for rifampicin resistance was confirmed by cloning and complementation in trans. Resistant isolates were characterized by multilocus sequence typing and PFGE. RESULTS: Between 1999 and 2008, 0.59% (96/16 348) of S. aureus clinical isolates studied showed rifampicin resistance. Rifampicin resistance was higher in methicillin-resistant S. aureus (MRSA) than in methicillin-susceptible S. aureus (MSSA) (3.26% versus 0.26%; P < 0.001). Twenty-two randomly selected rifampicin-resistant isolates were studied in depth, 11 showing low-level and 11 showing high-level rifampicin resistance (rifampicin MICs of 2-4 mg/L and ≥8 mg/L, respectively). Overall, 12 different mutations in the rpoB gene were detected, including a newly described N474K mutation followed by the insertion of a glycine residue at position 475. Among the eight different sequence types (STs) found, the most frequent were ST8 and ST863, the latter being associated with respiratory infections. Ten of the 11 low-level rifampicin-resistant isolates were MRSA ST8 and had the same H481N mutation, while the 11 high-level rifampicin-resistant isolates, 6 MSSA and 5 MRSA, belonged to eight different STs and had distinct rpoB mutations. CONCLUSIONS: Low-level rifampicin-resistant isolates were mainly clonal while high-level resistant isolates showed a high genetic diversity. Most mutations observed coincided with those found in other studies, but a new mutation conferring rifampicin resistance was detected.

In vitro activities of retapamulin and 16 other antimicrobial agents against recently obtained Streptococcus pyogenes isolates.

Retapamulin in vitro activity against 400 Streptococcus pyogenes clinical isolates obtained from skin, pharynx, ear fluid, and blood samples recovered from 2007 to 2009 was studied. The isolates belonged to 26 different emm types, including isolates nonsusceptible to erythromycin (n=187), tetracycline (n=99), ciprofloxacin (n=59), and bacitracin (n=43). Results were compared to the activities of 16 other antibiotics for topical and systemic use. Retapamulin MICs ranged from ≤0.015 to 0.12 µg/ml, showing the highest intrinsic activity among the topical antimicrobial drugs studied.

Spread of a highly mucoid Streptococcus pyogenes emm3/ST15 clone.

BACKGROUND: Hyaluronic acid capsule plays a key role in Streptococcus pyogenes virulence. Circulation of mucoid or highly encapsulated strains has been related to rheumatic fever epidemics and invasive disease in several countries. In 2009, an outbreak of mucoid S. pyogenes isolates was detected in northern Spain. The aim of the study was to describe clinical and molecular characteristics of mucoid strains causing this outbreak and to compare them with a sample of non-mucoid S. pyogenes isolates obtained during the same period of time. METHODS: All S. pyogenes isolates with a mucoid colony morphology (n = 132), 10% of non-mucoid (n = 144) and all invasive S. pyogenes isolates (n = 7) obtained in 2009 were included. Characterization was performed by T-agglutination, emm typing, pulsed field gel electrophoresis and multilocus sequence typing. RESULTS: One clone characterized as emm3.1/ST15 comprised 98.5% (n = 130) of all mucoid isolates. Subjects of all ages were affected. Main clinical manifestations were pharyngitis and scarlet fever, but this clone also caused invasive disease: two cases of streptococcal toxic shock syndrome, one arthritis, and one celullitis with a fatal outcome. Mucoid isolates were more prone to cause invasive disease than non-mucoid isolates (p = 0.001). CONCLUSIONS: Although no acute rheumatic fever cases were detected, the most worrisome characteristics of this clone were the success for causing invasive disease and the merge of two virulent features: the serotype, emm3, and capsule hyper-production, expressed as a mucoid morphology.

Phenotypic and genotypic characterization of Streptococcus pyogenes isolates displaying the MLSB phenotype of macrolide resistance in Spain, 1999 to 2005.

The aim of this study was to describe the genetic characteristics of Streptococcus pyogenes showing the MLSB phenotype of macrolide resistance from 1999 to 2005 in Spain and to highlight the substantial increase in these isolates in the last few years. The antimicrobial susceptibilities of 17,232 group A streptococci isolated from Madrid and Gipuzkoa from 1999 to 2005 were studied. The presence of the resistance genes ermA, ermB, mef, tetM, and tetO and the presence of the intTn and xis genes of the Tn916-Tn1545 transposon family were studied in a sample of 739 MLSB-resistant isolates. The epidemiological relationships among these isolates were analyzed by emm typing, T typing, and multilocus sequence typing. Erythromycin resistance was found in 21.3% of the isolates analyzed (annual variation of 14.3% to 28.9%). Until 2003, most erythromycin-resistant isolates showed the M phenotype, but in 2004 and 2005, about 50% of isolates showed the MLSB phenotype. Among the MLSB-resistant isolates studied, 16 clones were identified. The most prevalent clone was a strange emm11/T11/ST403 clone with a null yqiL allele. All but one of the 463 emm11/T11/ST403 isolates carried the ermB, tetM, intTn, and xis genes. The second most prevalent MLSB-resistant clone was emm28/T28/ST52, which comprised two subclones: one bacitracin-resistant, tetracycline-susceptible subclone carrying the ermB gene (n=115) and another bacitracin-susceptible, tetracycline-resistant subclone carrying the ermB and tetM genes (n=33). The rapid diffusion of these two clones, and especially of emm11/T11/ST403, caused the large increase in MLSB-resistant S. pyogenes isolates in Spain, suggesting a potential ability for international dissemination.

Erythromycin resistance and genetic elements carrying macrolide efflux genes in Streptococcus agalactiae.

The macrolide resistance determinants and genetic elements carrying the mef(A) and mef(E) subclasses of the mef gene were studied with Streptococcus agalactiae isolated in 2003 and 2004 from 7,084 vaginorectal cultures performed to detect carrier pregnant women. The prevalence of carriage was 18% (1,276 isolates), and that of erythromycin resistance 11.0% (129 of the 1,171 isolates studied). erm(B), erm(A) subclass erm(TR), and the mef gene, either subclass mef(A) or mef(E), were found in 72 (55.8%), 41 (31.8%), and 12 (9.3%) erythromycin-resistant isolates, while 4 isolates had more than 1 erythromycin resistance gene. Of the 13 M-phenotype mef-containing erythromycin-resistant S. agalactiae isolates, 11 had the mef(E) subclass gene alone, one had both the mef(E) and the erm(TR) subclass genes, and one had the mef(A) subclass gene. mef(E) subclass genes were associated with the carrying element mega in 10 of the 12 mef(E)-containing strains, while the single mef(A) subclass gene found was associated with the genetic element Tn1207.3. The nonconjugative nature of the mega element and the clonal diversity of mef(E)-containing strains determined by pulsed-field gel electrophoresis suggest that transformation is the main mechanism through which this resistance gene is acquired.

Yersinia enterocolitica O:3 infection of a prosthetic knee joint related to recurrent hemarthrosis.

Yersinia enterocolitica was isolated in joint fluid containing blood extracted from the knee of an 80-y-old woman with a 10-y history of total knee arthroplasty. Recurrent hemarthrosis had previously occurred in this knee. It appeared that the effusion of blood led to the deposition of iron on the joint, which may have contributed to the development of infection.