Distribution of macrolide resistance genes erm(B) and mef(A) among 160 penicillin-intermediate clinical isolates of Streptococcus pneumoniae isolated in southern France

Two prevalent mechanisms of macrolide resistance are currently described in pneumococci: production of rRNA methylase that modify 23S ribosomal RNA resulting in MLSB phenotype, and an active efflux system resulting in M-phenotype. These two mechanisms are mediated by erm(B) and mef(A) genes respectively. Several studies reported a predominance of mef(A) gene in United-States and Canada. In European countries, erm(B) determinant is prevalent and mef(A)-mediated erythromycin resistance was recently reported in about 10% of strains in Belgium and Italy. In order to evaluate implication of mef(A) gene in pneumococci erythromycin resistance, 160 clinical isolates of S. pneumoniae with low-level of penicillin resistance and resistance to macrolides recovered between April 1999 and April 2000 were collected. These isolates were tested for their macrolide susceptibility by disc diffusion method, 155 showed the MLSB phenotype and 5 the M phenotype. Genotypic analysis was performed by erm(B) and mef(A) specific-mediated PCR: erm(B) gene was detected in 154 isolates, mef(A) gene in 5 isolates, and both genes in one strain. The phenotype seems to be well correlated to the genotyping result except for strain harboring both resistance determinants. Molecular typing of isolates harboring mef(A) gene performed by pulsed-field gel electrophoresis (PFGE) after restriction by Smal shows these strains to be epidemiologically unrelated. Our results show the predominance of the erm(B) gene in erythromycin resistant S. pneumoniae isolates. mef(A)-mediated resistance is effective in Southern France (3.7%) but this rate is the lowest published from European countries.     

Molecular epidemiology of macrolide-resistant isolates of Streptococcus pneumoniae collected from blood and respiratory specimens in Norway

Norway has a low prevalence of antimicrobial resistance, including macrolide-resistant Streptococcus pneumoniae (MRSP). In a nationwide surveillance program, a total of 2,200 S. pneumoniae isolates were collected from blood cultures and respiratory tract specimens. Macrolide resistance was detected in 2.7%. M-type macrolide resistance was found in 60% of resistant isolates, and these were mainly mef(A)-positive, serotype-14 invasive isolates. The erm(B)-encoded macrolide-lincosamide-streptogramin B (MLS(B)) type dominated among the noninvasive isolates. One strain had an A2058G mutation in the 23S rRNA gene. Coresistance to other antibiotics was seen in 96% of the MLS(B)-type isolates, whereas 92% of the M-type isolates were susceptible to other commonly used antimicrobial agents. Serotypes 14, 6B, and 19F accounted for 84% of the macrolide-resistant isolates, with serotype 14 alone accounting for 67% of the invasive isolates. A total of 29 different sequence types (STs) were detected by multilocus sequence typing. Twelve STs were previously reported international resistant clones, and 75% of the macrolide-resistant isolates had STs identical or closely related to these clones. Eleven isolates displayed 10 novel STs, and 7/11 of these "Norwegian strains" coexpressed MLS(B) and tetracycline resistance, indicating the presence of Tn1545. The invasive serotype-14 isolates were all classified as ST9 or single-locus variants of this clone. ST9 is a mef-positive M-type clone, commonly known as England(14)-9, reported from several European countries. These observations suggest that the import of major international MRSP clones and the local spread of Tn1545 are the major mechanisms involved in the evolution and dissemination of MRSP in Norway.     

Identification of a new geographically widespread multiresistant Acinetobacter baumannii clone from European hospitals

The aim of the study was to investigate the genetic diversity of Acinetobacter baumannii clinical strains that had previously been allocated to three major groups based on automated ribotyping. Forty-seven isolates from European hospitals and one isolate from a South African hospital, geographically representative of the three ribogroups (ribogroups 1, 2 and 3 with 10, 23 and 15 isolates, respectively), were analysed using the highly discriminatory fingerprinting methods AFLP and pulsed-field gel electrophoresis (PFGE). Based on AFLP data, the isolates clustered into three main groups, each corresponding to one ribogroup. Inclusion of reference strains of the previously described clones I and II, responsible for outbreaks in northwestern European hospitals, showed that ribogroups 1 and 2 correspond to clones I and II, respectively, whereas ribogroup 3 apparently represents a new clone. This clone III was found in France, The Netherlands, Italy and Spain. Clones I and II were not limited to northwestern European countries, as they were also recovered from Spain, South Africa, Poland and Italy (clone I) and from Spain, Portugal, South Africa, France, Greece and Turkey (clone II). Combined AFLP and PFGE data showed intraclonal diversity and led to the distinction of 23 different genotypes. Three genotypes, two of them belonging to clone II and one to clone III, were found in different hospitals and may correspond to subsets of isolates with a more recent clonal relationship, which emphasizes the epidemic potential of these organisms.     

Genetic relationships among strains of avian Escherichia coli associated with swollen-head syndrome

Genetic diversity among 22 Escherichia coli strains isolated from chickens with swollen-head syndrome (SHS), an acute respiratory disease of domestic poultry, and 93 strains isolated from birds with colibacillosis was assessed on the basis of allelic variation at 20 enzyme-encoding loci detected by multilocus enzyme electrophoresis. SHS isolates from Spain and Canada were polymorphic at 14 loci and were classified into 19 multilocus genotypes, defining clones that differed on average at 34% of the loci. In most cases, SHS isolates of different clonal genotypes were distinct in O:H serotype and expressed different fimbrial antigens. Comparisons with 93 isolates obtained from birds with colibacillosis revealed enzyme polymorphisms at 17 of 20 loci, with an average of 3.5 alleles per locus. In the total sample, 56 clonal genotypes were distinguished, with 27 (23%) of the isolates belonging to one of three common clones. Both SHS and colibacillosis isolates were genetically diverse, with an average single-locus diversity of 0.36, indicating that a wide variety of naturally occurring bacterial clones is associated with these acute avian infections. Six previously defined groups of clones identified in diseased birds from the United States were represented in isolates from Spain, indicating that similar clones occur in widely separated geographic areas. In addition, one group of SHS isolates was closely related to a recognized widespread clone complex incriminated in human septicemia and meningitis. The results suggest that certain strains implicated in SHS infections belong to a clone complex whose members have special attributes that promote involvement in invasive diseases in humans and animals.     

Molecular typing indicates an important role for two international clonal complexes in dissemination of VIM-producing Pseudomonas aeruginosa clinical isolates in Hungary

VIM metallo-beta-lactamase-producing serotype O11 or O12 Pseudomonas aeruginosa isolates infecting or colonising 19 patients from seven hospitals were reported in Hungary between 2003 and 2005. In this study we characterised VIM-producing Pseudomonas spp. clinical isolates from two novel locations in Hungary; we identified three new bla(VIM) carrying integron types and the presence of the bla(VIM-2) allele in Hungary. By applying various typing techniques, including multilocus sequence typing, we revealed an important role of two international clonal complexes, CC4 and CC11, in the dissemination of bla(VIM)-positive P. aeruginosa in hospitals in Hungary. Isolate P12-Q, a representative strain from France of the major European multiresistant P12 clone, displayed ST111 which, according to eBURST analysis, is the presently calculated founder sequence type of CC4. This is in accordance with the wide geographic distribution of the P12 clone. Our data indicate that, although the CC4 clonal complex includes serotype O1 and O6 isolates as well, it also contains the P12 clone. We characterised a P. aeruginosa nosocomial clone with a singleton sequence type (ST313), that may have acquired bla(VIM-2) and bla(VIM-4) gene cassettes from a yet unidentified local gene pool in Hungary.     

Molecular epidemiology and distribution of serotypes, surface proteins, and antibiotic resistance among group B streptococci in Italy

Group B streptococci (GBS) comprising three different sets of isolates (31 invasive, 36 noninvasive, and 24 colonizing isolates) were collected in Italy during the years 2002 to 2005. Clonal groups were established by pulsed-field gel electrophoresis (PFGE), and selected isolates were studied by multilocus sequence typing (MLST). GBS isolates were also characterized by classical and molecular techniques for serotyping and protein gene and antibiotic resistance profiling. Some serotypes were significantly associated with a particular isolate population: serotype Ia more frequently corresponded to invasive strains than other strains, serotype V was more frequently encountered among noninvasive strains, and nontypeable strains were more common among isolates from carriers. Four major clonal groups accounted for 52.7% of all isolates: PFGE type 1/clonal complex 1 (CC1) comprised mainly serotype V isolates carrying the alp3 gene, PFGE type 2/CC23 encompassed serotype Ia isolates with the alp1 or alpha gene, PFGE type 3/CC17 comprised serotype III isolates carrying the rib gene, and PFGE type 4/CC19 consisted mainly of serotype II isolates possessing the rib gene. The same serotypes were shared by isolates of different clonal groups, and conversely, isolates belonging to the same clonal groups were found to be of different serotypes, presumably due to capsular switching by the horizontal transfer of capsular genes. Erythromycin resistance (prevalence, 16.5%; 15 resistant isolates of 91) was restricted to strains isolated from patients with noninvasive infections and carriers, while tetracycline resistance was evenly distributed (prevalence, 68.1%; 62 resistant isolates of 91). Most erythromycin-resistant GBS strains were of serotype V, were erm(B) positive, and belonged to the PFGE type 1/CC1 group, suggesting that macrolide resistance may have arisen both by clonal dissemination and by the horizontal transfer of resistance genes.     

Prevalence of serotypes and molecular epidemiology of Streptococcus pneumoniae strains isolated from children in Beijing, China: identification of two novel multiply-resistant clones

Three-hundred and seventy-six strains of Streptococcus pneumoniae isolated from clinical specimens and nasopharyngeal swabs from children at daycare centers and hospitals in Beijing China, between January 1997 and March 1998, were serotyped. Twenty-seven different serotypes were identified. The most prevalent serotypes in the carriage isolates were 6A, 19F, 23F, and 15 and were found in 66.8% of cases. Serotype data indicate that 51.8% of carrier strains would be included in the 11-valent conjugate vaccine formulation, while inclusion of vaccine-related serotypes, increased the potential vaccine coverage to 79.4%. Serotypes 7, 6B, 23F, 19F, 15, and 3 accounted for 62% of clinical strains, with 70% vaccine-related serotypes. DNA fingerprinting of 47 penicillin resistant and 71 penicillin-susceptible/macrolide-resistant strains by BOX polymerase chain reaction (PCR), pulsed-field gel electrophoresis (PFGE), and penicillin binding protein (PBP)-fingerprinting identified two novel clones: one a serotype 23F multiresistant clone resistant to penicillin, tetracycline, erythromycin, clindamycin, and variably resistant to chloramphenicol and trimethoprim-sulphamethoxazole; and the second a multiresistant penicillin-susceptible, macrolide-resistant serotype 6A clone, highly resistant also to tetracycline, clindamycin, and trimethoprim-sulphamethoxazole. The macrolide resistance determinant in 89% of erythromycin-resistant strains tested (penicillin-susceptible and penicillin-resistant) was the erm gene, both the erm and mef genes were simultaneously found in 6%, and mef alone in 3.4%. The data demonstrates that macrolide resistant strains in China include clonal strains and strains with dual mef and erm resistance determinants.     

Prevalence and molecular analysis of macrolide and fluoroquinolone resistance among isolates of Streptococcus pneumoniae collected during the 2000-2001 PROTEKT US Study

The PROTEKT US (Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin in the United States) surveillance program was established to determine the prevalence and mechanisms of antibacterial resistance among bacterial pathogens from patients with community-acquired respiratory tract infections. In year 1 of the PROTEKT US study, 10,103 isolates of Streptococcus pneumoniae, including 3,133 erythromycin-resistant strains and 81 levofloxacin-resistant strains, were collected from 206 centers. We report on the molecular analyses of these resistant strains. The resistance genotypes among the 3,044 typed macrolide-resistant isolates overall were mef(A) (n = 2,157; 70.9%), erm(B) (n = 530; 17.4%), mef(A) erm(B) (n = 304; 10.0%), and erm(A) subclass erm(TR) (n = 5; 0.2%). Fifty (1.6%) macrolide-resistant isolates were negative for the mef and the erm resistance genes. Seventy-eight (96.3%) of the 81 levofloxacin-resistant isolates analyzed possessed multiple mutations in the gyrA, gyrB, parC, and/or parE quinolone resistance-determining regions. A total of 43 known multilocus sequence typing (MLST) profiles (or single- or double-locus variants) accounted for 75 of 81 isolates. There was no evidence of dissemination of fluoroquinolone-resistant clones within the United States; however, 12 isolates with the same MLST profile were located in one center in Massachusetts. Almost 90% of the erythromycin-resistant isolates and approximately one-third of the levofloxacin-resistant isolates were multidrug resistant.     

Prevalence of erm methylase genes in clinical isolates of non-pigmented, rapidly growing mycobacteria

The aim of this study was to determine the frequency of erm genes coding for macrolide resistance among clinical isolates of non-pigmented rapidly growing mycobacteria (NPRGM) and to evaluate their importance in phenotypic resistance. Broth microdilution susceptibility testing was performed for all NPRGM tested. A PCR assay with consensus primers was used to evaluate the presence of erm genes among the 167 clinical isolates studied, which belonged to nine species of NPRGM; erm genes were detected in all nine species and 109 strains were erm -positive. The highest percentage of erm -positive isolates was found among Mycobacterium mageritense (100%) and the lowest among Mycobacterium mucogenicum (14%). The MICs of macrolides were found to be lower for erm -negative isolates (MIC₉₀: 2 mg/L) than for erm- positive isolates (MIC₉₀: 16 mg/L), although in some cases high MICs were found for erm -negative isolates. The finding that erm methylases are present in the majority of the species of NPRGM analysed in this study is not in agreement with conventional susceptibility studies. It therefore appears necessary to use a combination therapy to treat infections caused by NPRGM.     

Phenotypic and genotypic characterization of two penicillin-susceptible serotype 6B Streptococcus pneumoniae clones circulating in Italy

Twenty-nine penicillin-susceptible serotype 6B strains isolated from patients with invasive diseases and from healthy carriers were examined by different genotyping methods. Ten groups were identified on the basis of the pulsed-field gel electrophoresis (PFGE) profiles, and two of these contained multiple isolates and were analyzed further. PFGE group 1 comprised 12 isolates, the majority of which had a multiresistant phenotype (resistance to erythromycin, clindamycin, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole), corresponding to that of a clone previously described in the Mediterranean area and related to penicillin-resistant clone Spain(6B)-2. The pbp2b, pbp2x, dhf, and pspA genes of the isolates had identical restriction profiles; and the partial sequence of pspA was identical to that of clone Spain(6B)-2. In all isolates the resistance determinants erm(B) and tet(M) were inserted in a Tn1545-like element; 11 isolates carried cat as part of the integrated plasmid pC194. Multilocus sequence typing (MLST) performed with two isolates confirmed that their profiles corresponded to that of the Mediterranean clone. PFGE group 2 comprised nine strains, of which the majority showed no antibiotic resistance. Their pspA profiles were different, and the partial sequences obtained for two representative isolates indicated the presence of PspA proteins of different clades. The MLST profile of one strain was identical to that of a serotype 6B strain from the United Kingdom, while two other isolates were novel one-allele variants. This clone appears to be related (five of seven identical alleles) to two other internationally disseminated clones, Hungary(19A)-6 and Poland(23F)-16, both of which are penicillin resistant. The presence of antibiotic-susceptible isolates of this clone suggests that traits other than antibiotic resistance can make a clone successful.     

Rapid spread in Norway of an erythromycin-resistant pneumococcal clone, despite low usage of macrolides

During the last 4 years, Norway has experienced an increase in macrolide resistance among systemic isolates of Streptococcus pneumoniae. The Norwegian reference laboratory for pneumococci received the isolates from over 85% of the Norwegian cases of systemic pneumococcal disease in the period studied. To study the details of the increased macrolide resistance, all macrolide-resistant systemic pneumococcal isolates (410 isolates) collected in the period from 1995 to 2005 were characterized phenotypically, and a representative selection of 68 strains was also studied genotypically. The serogroups most frequently associated with macrolide resistance in the studied period were 14, 6, 23, 19, and 9. The resistance M-type was expressed in 85% of the resistant isolates. Of the 68 isolates analyzed by multilocus sequence typing, 19 different sequence types (STs) were represented, including several of the international resistant clones. All but one of the clones appeared at a low frequency; mainly as isolated cases. The increase in macrolide resistance seen from 2001 to 2005 proved to be caused by ST-9, defined as the England(14)-9 clone by the Pneumococcal Molecular Epidemiology Network. All ST-9 isolates tested, carried the mef(A) gene and expressed the resistance M-type. This clone first appeared in the Oslo region in 1993, but was by 2005 isolated from all over the country. Children were overrepresented among the cases caused by this clone; however, people aged 20-29, possibly involving the parent generation, were also represented at an increased frequency. The England(14)-9 clone has been able to spread successfully in the Norwegian population despite a relatively low consumption of macrolides.     

Molecular characterization of erythromycin-resistant clinical isolates of the four major antimicrobial-resistant Spanish clones of Streptococcus pneumoniae (Spain23F-1, Spain6B-2, Spain9V-3, and Spain14-5)

Erythromycin resistance and the characterization of the corresponding determinants of resistance were studied in clinical Streptococcus pneumoniae isolates belonging to the four major multiresistant pneumococcal Spanish clones (ermB and mefA genes for the Spain23F-1, Spain9V-3, serotype 14 variant of the Spain9V-3 and Spain14-5 clones and ermB gene for the Spain6B-2 clone). These isolates were confirmed as major clones by pulsed-field gel electrophoresis (PFGE), BOX-PCR, and multilocus sequence typing (MLST). The spread and prevalence of these erythromycin-resistant variants of the Spanish clones in an area of the north of Spain were dissimilar-low for the Spain9V-3 clone (5.8% among the isolates belonging to this clone, including isolates of the serotype 14 variant) and very frequent for the Spain14-5 clone (91.7%).     

Direct Detection of Macrolide Resistance in Mycoplasma genitalium Isolates from Clinical Specimens from France by Use of Real-Time PCR and Melting Curve Analysis

Mycoplasma genitalium is a sexually transmitted organism commonly treated with azithromycin. However, macrolide resistance has been reported and is associated with point mutations in the 23S rRNA gene. To evaluate the prevalence of macrolide resistance in M. genitalium isolates from clinical specimens from France, we first used a previously reported high-resolution melting assay. Because susceptible and resistant M. genitalium isolates were hardly discriminated in M. genitalium-positive clinical specimens, we developed a new molecular assay for the rapid detection of macrolide resistance. An assay using real-time PCR based on fluorescence resonance energy transfer (FRET) coupled with melting curve analysis was designed. The assay was first validated on characterized macrolide-resistant M. genitalium isolates and then applied to 202 urogenital M. genitalium-positive specimens collected from 178 patients from France in 2011 and 2012. Resistant genotypes were confirmed by 23S rRNA gene sequencing. Among the 202 M. genitalium-positive specimens, 155 were amplified, demonstrating a sensitivity of 76.7%. A substitution in the 23S rRNA gene was found in 14.2% of the patient samples. Nine and six patients had M. genitalium isolates with a substitution at positions 2059 and 2058, respectively. In four cases, a mixed population of wild-type and mutated M. genitalium isolates was observed. The prevalence of M. genitalium macrolide resistance has been stable in France since its detection in 2006. Our FRET PCR assay is able to discriminate between wild-type and resistant genotypes directly from clinical specimens. This assay will allow clinicians to shorten the time to the initiation of effective disease treatment.     

Molecular epidemiology of penicillin-susceptible non-beta-lactam-resistant Streptococcus pneumoniae isolates from Greek children

A total of 128 Streptococcus pneumoniae isolates that were susceptible to penicillin but resistant to non-beta-lactam agents were isolated from young carriers in Greece and analyzed by antibiotic susceptibility testing, serotyping, restriction fragment end labeling (RFEL), and antibiotic resistance genotyping. The serotypes 6A/B (49%), 14 (14%), 19A/F (11%), 11A (9%), 23A/F (4%), 15B/C (2%), and 21 (2%) were most prevalent in this collection. Of the isolates, 65% were erythromycin resistant, while the remaining isolates were tetracycline and/or trimethoprim-sulfamethoxazole resistant. Fifty-nine distinct RFEL types were identified. Twenty different RFEL clusters, harboring 2 to 19 strains each, accounted for 76% of all strains. Confirmatory multilocus sequence typing analysis of the genetic clusters showed the presence of three international clones (Tennessee(23F)-4, England(14)-9, and Greece(6B)-22) representing 30% of the isolates. The erm(B) gene was present in 70% of the erythromycin-resistant isolates, whereas 18 and 8% contained the mef(A) and mef(E) genes, respectively. The pneumococci representing erm(B), erm(A), and mef genes belonged to distinct genetic clusters. In total, 45% of all isolates were tetracycline resistant. Ninety-six percent of these isolates contained the tet(M) gene. In conclusion, penicillin-susceptible pneumococci resistant to non-beta-lactams are a genetically heterogeneous group displaying a variety of genotypes, resistance markers, and serotypes. This suggests that multiple genetic events lead to non-beta-lactam-resistant pneumococci in Greece. Importantly, most of these genotypes are capable of disseminating within the community.     

Delineation of the endemic and sporadic methicillin-resistant Staphylococcus aureus clones in a Czech hospital

The aim of this study was to define the endemic clones of methicillin-resistant Staphylococcus aureus (MRSA) among strains collected between September, 2001, and February, 2003, at the regional hospital of Novy Jicín, Czech Republic. The isolates were characterized by susceptibility tests, HindIII ribotyping, and pulsed-field gel electrophoresis. Representatives of each clonal type were analyzed by multilocus sequence typing and staphylococcal cassette chromosome mec (SCCmec) typing. The prevalence of the most important macrolide (ermA, ermB, ermC, and msrA) and aminoglycoside (aac6'-aph2", aph3', and ant4') resistance genes was evaluated as well. Our results document the existence of two international MRSA clones: (1) the Iberian clone (ST247:SCCmec IA:PFGE A:ribotype H2), endemic in the hospital and associated to a single multiresistant phenotype; and (2) clone EMRSA-15 (ST22:SCCmec IV:PFGE H-ribotype H7), appearing in the beginning of 2002 and associated with three phenotypes. These two clones could be distinguished by antibiogram, distribution of macrolide and aminoglycoside resistance genes (ermA, aac6'-aph2", ant4' versus ermC and msrA in a few isolates), production of beta-lactamase, and presence of enterotoxin A (in the Iberian clone).     

Phenotypes, genotypes, serotypes and molecular epidemiology of erythromycin-resistant Streptococcus agalactiae in Italy

The purpose of this investigation was to analyse Streptococcus agalactiae (group B Streptococcus, GBS) isolates collected in Italy from vaginal and urine samples in respect to their clonality, distribution of virulence factors and antimicrobial resistance determinants. Three hundred and eighty-eight GBS were recovered from clinical samples. They were analysed for antibiotic resistance profiling. Erythromycin-resistant strains were further characterised by multilocus sequence typing (MLST), serotyping and the detection of alp genes of the alpha-like protein (Alp) family. GBS isolates represented 40 different sequence types (STs), grouped in five clonal complexes (CCs) and belonged to seven serotypes. Most serotype V strains (81%) possessed alp2-3; serotype Ia carried mainly epsilon, while the serotype III mainly rib. All isolates were susceptible to penicillin, whereas resistance to erythromycin was detected in 15% of isolates. Most erythromycin-resistant GBS strains were of serotype V (56.8%) and belonged to the CC-1 group (50%). Macrolide resistance phenotypes were the cMLS(B) (46.5%) and the M phenotypes (46.5%) due to the presence of ermB and mefA/E genes, respectively. These results provide data which establish a baseline for monitoring erythromycin resistance in this region and also provide an insight into the correlation among clonal types, serotypes, surface protein and resistance genes. The increased prevalence of strains that displayed the M phenotype strengthens the importance of the epidemiological surveillance of macrolide resistance in GBS, which may also represent an important reservoir of resistance genes for other species.     

Clonality behind the increase of multidrug-resistance among non-invasive pneumococci in Southern Finland

Multidrug-resistance among Streptococcus pneumoniae isolates, especially of serotype 19A, has increased in several countries recently. Even before the introduction of the pneumococcal conjugate vaccine into the Finnish National Vaccination Programme, the proportion of multidrug-resistant (MDR) pneumococci had doubled from 2007 to 2008, when it reached 3.6% in Southern Finland. Our aim was to look for a possible association between antimicrobial susceptibility and clonality among the MDR isolates. Twelve non-invasive isolates non-susceptible to penicillin, erythromycin, clindamycin, trimethoprim/sulfamethoxazole, and doxycycline from 2008 were available for serotyping, genotyping by multilocus sequence typing (MLST), and detection of genes encoding macrolide resistance and adherence-promoting pili. Two isolates were also resistant to ceftriaxone. Five serotypes, 19F, 19A, 6B, 23F, and 14, and six genotypes from three genetic lineages were found, among which CC320 was the largest. All isolates in this study carried the erm(B) macrolide resistance gene, and the CC320 isolates additionally carried the mef(A/E) macrolide resistance gene. Eleven isolates carried pilus islet 1, while the CC320 isolates also carried the pilus islet 2 genes. The findings emphasize the importance of the careful monitoring of antimicrobial susceptibility and serotype distribution among pneumococci, especially now that antimicrobials and pneumococcal vaccines are in widespread use.     

A multiresistant clone of Pseudomonas aeruginosa sequence type 773 spreading in a burn unit in Orumieh,Iran

Herein, we describe the phenotypic and genotypic characterization of a multiresistant clone of Pseudomonas aeruginosa disseminating in a burn unit in Orumieh, Iran. A total of 58 isolates of P. aeruginosa were collected during August 2007 and June 2008. Minimum inhibitory concentrations (MICs) of P. aeruginosa isolates were determined against 11 antimicrobial agents by E test. Serotyping, pulsed‐field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) were used for studying the clonal relationship among the isolates. Antibiotic susceptibility testing revealed that most of the isolates were multidrug resistant and colistin was the antibiotic with the highest activity. Pseudomonas aeruginosa isolates fell into nine different serotypes, and O10 and O11 were the most common. PFGE analyses showed 12 different genotypes and 68.1% of isolates showed more than 80% similarity, indicating possible clonal relatedness. These isolates were found to belong to the same sequence type, ST773. This sequence type has earlier been reported from China, and a double locus variant of this ST has been found earlier in France in a PER‐1 extended‐spectrum β‐lactamase‐producing P. aeruginosa.     

A common clone of erythromycin-resistant Streptococcus pneumoniae in Greece and the UK

Objective  To investigate the possible genetic relationship among erythromycin-resistant Streptococcus pneumoniae strains isolated in Greece and the UK.
Methods  During 1995–97, 140 S. pneumoniae strains were isolated from clinical specimens submitted to the microbiology departments of the two main children's hospital in Athens. All erythromycin-resistant strains were further studied with respect to the presence of genes encoding for the two major mechanisms of macrolide resistance, their serotypes, and pulsed-field gel electrophoresis (PFGE) types, in comparison to a previously characterized UK erythromycin-resistant clone.
Results  Eleven of the 140 isolates (7.9%) were resistant to erythromycin; nine of these were susceptible to penicillin. Serotyping allocated seven, three and one isolates to serotypes 14, 19F and serogroup 6, respectively. The mef A gene was detected in seven isolates (five serotype 14 and two serotype 19F), erm B in two (one serotype 19F and the serogroup 6 isolate), whilst in the remaining two isolates no resistance gene could be detected by polymerase chain reaction (PCR). Pulsed-field gel electrophoresis of genomic DNA showed that five Greek serotype 14 isolates belonged to the same chromosomal type as the serotype 14 erythromycin-resistant UK clone.
Conclusions  The present study showed that erythromycin resistance among the S. pneumoniae isolates was mostly owing to the efflux mechanism and suggested a possible clonal spread of serotype 14 erythromycin-resistant S. pneumoniae strains between Greece and the UK.     

Identification of macrolide-resistant clones of Streptococcus pyogenes in Portugal

Although the overall level of macrolide resistance (27%) has remained stable in Portugal, a rapid inversion in the dominant phenotypes has been noted, with a sharp decrease in the MLS(B) phenotype paralleled by an increase in the M phenotype. To gain further insight into these changes, 325 macrolide-resistant isolates were characterised using a combination of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The use of Cfr9I, an isoschizomer of SmaI, to digest M phenotype isolates that were refractory to SmaI digestion allowed direct comparison of MLS(B) and M isolates. The results from PFGE and MLST were highly concordant and identified eight major clones, accounting for 92% of the isolates, each of which was associated exclusively with a single macrolide resistance phenotype. Two major clones were found among MLS(B) isolates, characterised by sequence types (ST) 46 (T12/emm22) and ST52 (T28/emm28), whereas clones characterised by ST39 (T4/emm4) and ST28 (T1/emm1) dominated among M isolates. The clone defined by ST52 corresponded to a bacitracin-resistant clone circulating in Europe, and a novel variant expressing other surface antigens (T12/emm22) was detected. The presence of the four major clones has been reported previously in other European countries, suggesting Europe-wide dissemination of a few macrolide-resistant lineages.