Epidemiological and molecular characterization of Streptococcus pneumoniae carriage strains in pre-school children in Arkhangelsk, northern European Russia, prior to the introduction of conjugate pneumococcal vaccines.

BACKGROUND: The 13-valent Pneumococcal Conjugate Vaccine (PCV-13) was introduced in the National Immunization Programme (NIP) schedule in Russia in March 2014. Previously, the 7-valent Pneumococcal Conjugate Vaccine (PCV-7) was marketed in Russia in 2009 but has never been offered for mass vaccination. A carriage study was performed among children in Arkhangelsk in 2006. The objective was to determine the prevalence of carriage, serotype distribution, antimicrobial susceptibility and the molecular structure of Streptococcus pneumoniae strains before marketing and introduction of PCV-13. METHODS: A cross-sectional study was conducted on a cluster-randomized sample of children and a self-administrated questionnaire for parents/guardians.  Nasopharyngeal samples were collected from 438 children younger than 7 years attending nurseries and kindergartens in the Arkhangelsk region, Russia. Detailed demographic data, as well as information about the child's health, traveling, exposure to antimicrobials within the last 3 months and anthropometric measurements were collected for all study subjects. Variables extracted from the questionnaire were analysed using statistic regression models to estimate the risk of carriage. All pneumococcal  isolates were examined with susceptibility testing, serotyping and multilocus sequence typing. RESULTS: The overall prevalence of asymptomatic carriage was high and peaking at 36 months with a rate of 57%. PCV-13 covered 67.3% of the detected strains. High rates of non-susceptibility to penicillin, macrolides and multidrug resistance were associated with specific vaccine serotypes, pandemic clones, and local sequence types. Nine percent of isolates represented three globally disseminated disease-associated pandemic clones; penicillin- and macrolide-resistant clones NorwayNT-42 and Poland6B-20, as well as penicillin- and macrolide-susceptible clone Netherlands3-31. A high level of antimicrobial consumption was noted by the study. According to the parent's reports, 89.5% of the children used at least one antimicrobial regime since birth. None of the hypothesised predictors of S. pneumoniae carriage were statistically significant in univariable and multivariable logistic models. CONCLUSIONS: The study identified a high coverage of the PCV-13-vaccine, but serotype replacement and expansion of globally disseminated disease-associated clones with non-vaccine serotypes may be expected. Further surveillance of antimicrobial resistance and serotype distribution is therefore required.

Faecal colonization of E. coli and Klebsiella spp. producing extended-spectrum beta-lactamases and plasmid-mediated AmpC in Mozambican university students.

BACKGROUND: In recent years, the world has seen a surge in Enterobacteriaceae resistant to broad-spectrum beta-lactam antibiotics due to the production of extended-spectrum beta-lactamases (ESBLs) or plasmid-mediated AmpC (pAmpC) enzymes. Data on the epidemiology of cephalosporin-resistant Enterobacteriaceae in Sub-Saharan Africa are still limited. METHODS: Two hundred seventy-five non-repetitive stool samples were collected from Mozambican university students of both sexes. Samples were cultured on MacConkey agar with and without ceftriaxone (1 mg/L) for selection of third-generation cephalosporin-resistant isolates, which were subjected to antimicrobial susceptibility testing by disc diffusion, characterization of resistance genes by PCR and ERIC-PCR analysis for strain clonality. RESULTS: Among the 275 students, 55 (20%) carried a total of 56 E. coli (n = 35) and Klebsiella spp. (n = 21) isolates resistant to ceftriaxone and phenotypically positive for ESBL- and/or pAmpC-production. Forty-three percent of the isolates (24/56) contained only ESBL genes, 11% (6/56) only pAmpC genes, and 36% (20/56) both ESBL and pAmpC genes. The remaining six isolates were negative for the CTX-M/pAmpC genes included in the test panel. E. coli and Klebsiella spp. combined demonstrated 70% resistance to tetracycline and co-trimoxazole, 63% to ceftazidime and 34% to ciprofloxacin. In total, 89% of ESBL/pAmpC-positive isolates were defined as multi-resistant by being resistant to three or more antibiotic classes. ERIC-PCR fingerprinting demonstrated low similarity among isolates. None of the participants reported recent hospitalization and just 12.5% had taken antibiotics 3 months prior to the study. CONCLUSION: This study demonstrated 20% colonization with multi-resistant E. coli and Klebsiella spp. among Mozambican students with a diversity of ESBL and pAmpC genes. Colonization was not related to prior hospitalization or antimicrobial consumption.

Extended-spectrum ß-lactamase-producing Enterobacteriaceae among pregnant women in Norway: prevalence and maternal-neonatal transmission.

OBJECTIVE: To study (i) the prevalence and risk factors for carriage of extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-E) in pregnant women, (ii) the maternal-neonatal transmission rate of ESBL-E at birth and (iii) the prevalence of ESBL-E in expressed breast milk of colonized mothers. STUDY DESIGN: In this cross-sectional, population-based study with case follow-up on maternal-neonatal transmission of ESBL-E, women were screened for rectal ESBL-E colonization at 36 weeks of pregnancy and delivery. Possible risk factors for colonization were studied by logistic regression. Infants of ESBL-E-positive mothers were screened for ESBL-E during their first weeks of life. ESBL-encoding genes were detected by PCR and clonal relatedness was investigated by pulsed-field gel electrophoreses. RESULTS: In total, 26 out of 901 (2.9%) women were colonized by ESBL-producing Escherichia coli at 36 weeks of pregnancy. One of the women carried an additional ESBL Klebsiella pneumoniae strain. Adjusted for traveling, African or Asian nationality was a risk factor for colonization; OR=5.62 (2.21, 14.27) (LR-p=0.003). Fourteen women remained ESBL-E carriers at delivery. ESBL-E strains indistinguishable from the strains isolated from their respective mothers were detected in 5 (35.7%) infants during their first days of life (median day 3; range=2 to 8). A total of 146 expressed milk samples were cultured from 25 out of 26 colonized mothers, all were ESBL-E negative. CONCLUSIONS: The prevalence of ESBL-E carriage among pregnant women was low in our region, but the high maternal-neonatal transmission rate suggests that colonized mothers represent a substantial risk for infant colonization.

Emergence of clonally related multidrug resistant Haemophilus influenzae with penicillin-binding protein 3-mediated resistance to extended-spectrum cephalosporins, Norway, 2006 to 2013.

Resistance to cephalosporins in Haemophilus influenzae is usually caused by characteristic alterations in penicillin-binding protein 3 (PBP3), encoded by the ftsI gene. Resistance to extended-spectrum cephalosporins is associated with high-level PBP3-mediated resistance (high-rPBP3), defined by the second stage S385T substitution in addition to a first stage substitution (R517H or N526K). The third stage L389F substitution is present in some high-rPBP3 strains. High-rPBP3 H. influenzae are considered rare outside Japan and Korea. In this study, 30 high-rPBP3 isolates from Norway, collected between 2006 and 2013, were examined by serotyping, multilocus sequence typing (MLST), ftsI sequencing, detection of beta-lactamase genes and minimum inhibitory concentration (MIC) determination. MICs were interpreted according to clinical breakpoints from the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Respiratory isolates predominated (proportion: 24/30). The 30 isolates included one serotype f isolate, while the remaining 29 lacked polysaccharide capsule genes. Resistance to extended-spectrum cephalosporins (cefixime, 29 isolates/30 isolates; cefepime, 28/30; cefotaxime, 26 /30; ceftaroline, 26/30; ceftriaxone, 14/30), beta-lactamase production (11/30) and co-resistance to non-beta-lactams (trimethoprim-sulfamethoxazole, 13/30; tetracycline, 4/30; chloramphenicol, 4/30; ciprofloxacin, 3/30) was frequent. The N526K substitution in PBP3 was present in 23 of 30 isolates; these included a blood isolate which represents the first invasive S385T + N526K isolate reported from Europe. The L389F substitution, present in 16 of 30 isolates, coincided with higher beta-lactam MICs. Non-susceptibility to meropenem was frequent in S385T + L389F + N526K isolates (8/12). All 11 beta-lactamase positive isolates were TEM-1. Five clonal groups of two to 10 isolates with identical MLST-ftsI allelic profiles were observed, including the first reported high-rPBP3 clone with TEM-1 beta-lactamase and co-resistance to ciprofloxacin, tetracycline, chloramphenicol and trimethoprim-sulfamethoxazole. Prior to this study, no multidrug resistant high-rPBP3 H. influenzae had been reported in Norway. Intensified surveillance of antimicrobial resistance is needed to guide empiric therapy.

Large IncHI2-plasmids encode extended-spectrum ß-lactamases (ESBLs) in Enterobacter spp. bloodstream isolates, and support ESBL-transfer to Escherichia coli.

We investigated the prevalence of extended-spectrum ß-lactamases (ESBLs) in Enterobacter spp. bloodstream isolates from 19 hospital laboratories in Norway during 2011. A total of 62/230 (27%) isolates were resistant to third-generation cephalosporins and four (1.7%) were ESBL-positive; blaCTX -M-15 (n = 3) and blaSHV -12 (n = 1). This is comparable to the prevalence of ESBLs in clinical isolates of Escherichia coli and Klebsiella pneumoniae in Norway during the same period. All ESBL-positive isolates were multidrug resistant (MDR) and harboured plasmid-mediated quinolone resistance. Three isolates supported transfer of large IncHI2-plasmids harbouring ESBL- and MDR-encoding genes to E. coli recipients by in vitro conjugation.

Prevalence and population structure of Staphylococcus aureus nasal carriage in healthcare workers in a general population. The Tromsø Staph and Skin Study.

Healthcare workers (HCWs) may be a reservoir for Staphylococcus aureus transmission to patients. We examined whether HCW status is associated with S. aureus nasal carriage and population structure (spa types) in 1302 women (334 HCWs) and 977 men (71 HCWs) aged 30-69 years participating in the population-based Tromsø Study in 2007-2008. Multivariable logistic regression models were used. While no methicillin-resistant S. aureus (MRSA) was isolated, overall, 26·2% of HCWs and 26·0% of non-HCWs were S. aureus nasal carriers. For women overall and women residing with children, the odds ratios for nasal carriage were 1·54 [95% confidence interval (CI) 1·09-2·19] and 1·86 (95% CI 1·14-3·04), respectively, in HCWs compared to non-HCWs. Moreover, HCWs vs. non-HCWs had a 2·17 and 3·16 times higher risk of spa types t012 and t015, respectively. This supports the view that HCWs have an increased risk of S. aureus nasal carriage depending on gender, family status and spa type.

Enterococcus faecalis from patients with chronic periodontitis: virulence and antimicrobial resistance traits and determinants.

This study investigated the presence of virulence and resistance traits, as well as their genetic determinants in subgingival Enterococcus faecalis from patients with chronic periodontitis. Twenty-four E. faecalis strains from a previously multi-locus sequence typing (MLST)-characterized strain collection were examined for virulence-associated phenotypes, antimicrobial susceptibility, and virulence- and antimicrobial-resistant determinants. Gelatinase, hemolysin, and biofilm production were detected in 50, 17, and 100% of the strains, respectively. Genes encoding adherence factors such as ace, efaA, and bopD were detected in all isolates. Other putative virulence determinants, i.e., EF3314, gelE, asa, esp, cylA, ef1841/fsrC, and asa373, were found in a portion of the strains. Different levels of resistance were observed in these strains, with two strains expressing high-level resistance to erythromycin and gentamicin. The integrase gene and accessory gene(s) of the Tn916/Tn1545 family were detected in ten strains. A direct link was shown between the presence of Tn916/Tn1545-like elements and resistance to doxycycline and/or erythromycin. The results demonstrated that virulence and antibiotic resistance determinants were prevalent in oral E. faecalis strains. It implicates that oral E. faecalis might play a role in the pathogenesis of chronic periodontitis and be a potential reservoir for the transferable elements of virulence and antimicrobial resistance.

Molecular characterization of VIM-producing Klebsiella pneumoniae from Scandinavia reveals genetic relatedness with international clonal complexes encoding transferable multidrug resistance.

VIM-producing Klebsiella pneumoniae (VPKP) has been identified as a source of hospital outbreaks and is prevalent particularly in the Mediterranean region. In this study we have characterized eight VPKP isolates identified in Scandinavia during 2005-2008. With the exception of one isolate, all were from patients with recent history of hospitalization abroad (Greece, n = 6; Turkey, n = 1). Multilocus sequence typing (MLST) resulted in five sequence types (STs), ST36 (n = 1), ST147 (n = 4), ST272 (n = 1), ST273 (n = 1) and ST383 (n = 1), which except for ST272 were part of putative international clonal complexes. All were multidrug resistant due to the presence of other resistance determinants, including extended-spectrum ß-lactamases (CTX-M-3, SHV-5 and SHV-12), 16S rRNA methylases (ArmA) and plasmid-mediated quinolone resistance determinants (QnrS). One isolate harboured a novel VIM-variant (VIM-26) while VIM-1 and VIM-19 were detected in six and one isolate, respectively. Two different genetic structures surrounding the bla(VIM) gene were identified in four isolates. In two isolates bla(VIM-1) and bla(VIM-26) were located in an integron similar to In-e541 (intI1;bla(VIM-1/-26);aacA7; dhfrI;aadA1;3'CS) while in the other two isolates bla(VIM-1) was located in an integron lacking 3'CS but with an IS26 element in the 3'end (intI1;bla(VIM-1);aac(6')-Ib;IS26), as identified in the IncN plasmid pKOX105. The bla(VIM) -genes were located on transferable plasmids ranging from ∼40 to ∼240 kb and associated with Tn21 in four isolates. PCR-based replicon typing indicated association of bla(VIM) with IncN (n = 3) and A/C (n = 1) broad-host-range plasmids but also with unknown replicons (n = 4). In conclusion, Scandinavian VPKP is associated with importation and genetically related to international clones encoding transferable plasmid-mediated multidrug resistance.

Retrospective evidence for a biological cost of vancomycin resistance determinants in the absence of glycopeptide selective pressures.

OBJECTIVES: To estimate the relative fitness differences between glycopeptide-resistant Enterococcus faecium (GREF) and glycopeptide-susceptible E. faecium (GSEF) from yearly surveillance data on the occurrence of GREF in Danish poultry farm environments. METHODS: A population genetic model was adapted to retrospectively estimate the biological fitness cost of acquired resistance. Maximization of a likelihood function was used to predict the longitudinal persistence of acquired resistance. RESULTS: Our analysis suggests strong selection against GREF following the 1995 ban on the glycopeptide growth promoter avoparcin. However, parameterizing the model with two selection coefficients suggesting a reduced negative effect of the acquired resistance on bacterial fitness over time significantly improved the fit of the model. Our analyses suggest that the acquired glycopeptide resistance will persist for >25 years. CONCLUSIONS: Acquired resistance determinants in commensal E. faecium populations in Danish farm environments are likely to persist for decades, even in the absence of glycopeptide use.

A sensitive and specific phenotypic assay for detection of metallo-ß-lactamases and KPC in Klebsiella pneumoniae with the use of meropenem disks supplemented with aminophenylboronic acid, dipicolinic acid and cloxacillin.

Enterobacteriaceae producing carbapenemases, such as KPC or metallo-ß-lactamases (MBLs), have emerged on several continents. Phenotypic tests are urgently needed for their rapid and accurate detection. A novel carbapenemase detection test, comprising a meropenem disk, and meropenem disks supplemented with 730 µg of EDTA, 1000 µg of dipicolinic acid (DPA), 600 µg of aminophenylboronic acid (APBA), or 750 µg of cloxacillin, was evaluated against Klebsiella pneumoniae isolates with KPC (n = 34), VIM (n = 21), IMP (n = 4) or OXA-48 (n = 9) carbapenemases, and carbapenem-resistant Enterobacteriaceae with porin loss in combination with an extended-spectrum ß-lactamase (ESBL) (n = 9) or AmpC hyperproduction (n = 5). Commercially available diagnostics tablets from Rosco containing meropenem and the same inhibitors as described above (except EDTA) were also evaluated. An increased meropenem inhibition zone was sought in the presence of each added ß-lactamase inhibitor. APBA had excellent sensitivity for detecting K. pneumoniae with KPC enzymes. Isolates with combined AmpC hyperproduction and porin loss were also positive in the APBA test but, unlike KPC producers, showed cloxacillin synergy. Both DPA and EDTA had excellent sensitivity for detection of MBL-producing K. pneumoniae. However, EDTA showed poor specificity, with positive results noted for 1/9 ESBL-producing isolates, for 4/34 KPC-producing isolates, and for 4/9 OXA-48-producing isolates, whereas all of these were negative when DPA was used. The in-house test distinguished accurately between several different mechanisms mediating reduced susceptibility to carbapenems in Enterobacteriaceae. The commercial combination tablets from Rosco performed similarly to the in-house test, with the exception of one false-positive MBL result and one false-positive KPC result among the OXA-48 producers.

Comparison of disk diffusion, Etest and VITEK2 for detection of carbapenemase-producing Klebsiella pneumoniae with the EUCAST and CLSI breakpoint systems.

The aim of this study was to compare CLSI and EUCAST MIC and disk diffusion carbapenem breakpoints for the detection of carbapenemase-producing Klebsiella pneumoniae. K. pneumoniae strains with known KPC (n = 31) or VIM (n = 20) carbapenemases were characterized by disk diffusion (Oxoid) and Etest (bioMérieux) vs. imipenem, meropenem and ertapenem, and with VITEK2 (bioMérieux, five different cards). Extended-spectrum ß-lactamase (ESBL) testing was performed with VITEK2 (bioMérieux), ESBL combination disks (Becton Dickinson) and the ESBL Etest (bioMérieux). With CLSI and EUCAST MIC breakpoints, respectively, 11 and seven of the strains were susceptible to imipenem, 12 and eight to meropenem, and seven and none to ertapenem. The EUCAST epidemiological cut-off (ECOFF) values for meropenem and ertapenem identified all carbapenemase producers, whereas the imipenem ECOFF failed in five strains. All carbapenemase producers were detected with EUCAST disk diffusion breakpoints for ertapenem and meropenem, and four strains were susceptible to imipenem. CLSI disk diffusion breakpoints characterized 18 (imipenem), 14 (meropenem) and three (ertapenem) isolates as susceptible. When cards with a single carbapenem were used, detection failures with VITEK2 were four for imipenem, none for meropenem and one for ertapenem. Cards containing all three carbapenems had one to two failures. With ESBL combination disks, 21/31 KPC producers and 2/20 VIM producers were positive. With VITEK2, no VIM producers and between none and seven KPC producers were ESBL-positive. All carbapenemase producers were detected with the meropenem MIC ECOFF, or the clinical EUCAST breakpoint for ertapenem. EUCAST disk diffusion breakpoints for meropenem and ertapenem detected all carbapenemase producers. VITEK2 had between none and four failures in detecting carbapenemase producers, depending on the antibiotic card.

Mobile genetic elements and their contribution to the emergence of antimicrobial resistant Enterococcus faecalis and Enterococcus faecium.

Mobile genetic elements (MGEs) including plasmids and transposons are pivotal in the dissemination and persistence of antimicrobial resistance in Enterococcus faecalis and Enterococcus faecium. Enterococcal MGEs have also been shown to be able to transfer resistance determinants to more pathogenic bacteria such as Staphylococcus aureus. Despite their importance, we have a limited knowledge about the prevalence, distribution and genetic content of specific MGEs in enterococcal populations. Molecular epidemiological studies of enterococcal MGEs have been hampered by the lack of standardized molecular typing methods and relevant genome information. This review focuses on recent developments in the detection of MGEs and their contribution to the spread of antimicrobial resistance in clinically relevant enterococci.

Tn1546 is part of a larger plasmid-encoded genetic unit horizontally disseminated among clonal Enterococcus faecium lineages.

OBJECTIVES: To determine the genetic composition of the first VanA-type plasmid (pIP816) reported, which was isolated from a clinical Enterococcus faecium (BM4147) strain in France in 1986, and to reveal the genetic units responsible for the dissemination of the vanA gene cluster by comparisons with current, published and additionally generated vanA-spanning plasmid sequences obtained from a heterogeneous E. faecium strain collection (n = 28). METHODS: Plasmid sequences were produced by shotgun sequencing using ABI dye chemistry and primer walking, and were subsequently annotated. Comparative sequence analysis of the vanA region was done with published plasmids, with a partial vanA plasmid (pVEF4) reported here and to >140 kb of sequence obtained from a collection of vanA-harbouring plasmid fragments. RESULTS: Bioinformatic analyses revealed that pIP816 from 1986 and contemporary vanA plasmids shared a conserved genetic fragment of 25 kb, spanning the 10.85 kb vanA cluster encoded by Tn1546, and that the larger unit is present in both clinical and animal complexes of E. faecium. A new group II intron in pVEF4 was characterized. CONCLUSIONS: Comparative DNA analyses suggest that Tn1546 disseminates in and between clonal complexes of E. faecium as part of a larger genetic unit, possibly as a composite transposon flanked by IS1216 elements.

Sporadic occurrence of CMY-2-producing multidrug-resistant Escherichia coli of ST-complexes 38 and 448, and ST131 in Norway.

Clinical isolates of Escherichia coli with reduced susceptibility to oxyimino-cephalosporins and not susceptible to clavulanic acid synergy (n = 402), collected from Norwegian diagnostic laboratories in 2003-2007, were examined for the presence of plasmid-mediated AmpC beta-lactamases (PABLs). Antimicrobial susceptibility testing was performed for beta-lactam and non-beta-lactam antibiotics using Etest and Vitek2, respectively. The AmpC phenotype was confirmed using the boronic acid test. PABL-producing isolates were detected using ampC multiplex-PCR and examined by bla(AmpC) sequencing, characterization of the bla(AmpC) genetic environment, phylogenetic grouping, XbaI- pulsed-field gel electrophoresis (PFGE), multi-locus sequence typed (MLST), plasmid profiling and PCR-based replicon typing. For the PABL-positive isolates (n = 38), carrying bla(CMY-2) (n = 35), bla(CMY-7) (n = 1) and bla(DHA-1) (n = 2), from out- (n = 23) and in-patients (n = 15), moderate-high MICs of beta-lactams, except cefepime and carbapenems, were determined. All isolates were resistant to trimethoprim-sulphamethoxazole. Multidrug resistance was detected in 58% of the isolates. The genes bla(CMY-2) and bla(CMY-7) were linked to ISEcp1 upstream in 32 cases and in one case, respectively, and bla(DHA-1) was linked to qacEDelta1sul1 upstream and downstream in one case. Twenty isolates were of phylogenetic groups B2 or D. Thirty-three XbaI-PFGE types, including three clusters, were observed. Twenty-five sequence types (ST) were identified, of which ST complexes (STC) 38 (n = 7), STC 448 (n = 5) and ST131 (n = 4) were dominant. Plasmid profiling revealed 1-4 plasmids (50-250 kb) per isolate and 11 different replicons in 37/38 isolates; bla(CMY-2) was carried on transferable multiple-replicon plasmids, predominantly of Inc groups I1 (n = 12), FII (n = 10) and A/C (n = 7). Chromosomal integration was observed for bla(CMY-2) in ten strains. CMY-2 is the dominant PABL type in Norway and is associated with ISEcp1 and transferable, multiple-replicon IncI1, IncA/C, or IncFII plasmids in nationwide strains of STC 448, STC 38 and ST131.

Complete sequence of Enterococcus faecium pVEF3 and the detection of an omega-epsilon-zeta toxin-antitoxin module and an ABC transporter.

Glycopeptide resistant Enterococcus faecium (GREF) persists on Norwegian poultry farms despite the ban on the growth promoter avoparcin. The biological basis for long-term persistence of avoparcin resistance is not fully understood. This study presents the complete DNA sequence of the E. faecium R-plasmid pVEF3 and functional studies of some plasmid-encoded traits (a toxin-antitoxin (TA) system and an ABC transporter) that may be of importance for plasmid persistence. The pVEF3 (63.1 kbp), isolated from an E. faecium strain of poultry origin sampled in Norway in 1999, has 71 coding sequences including the vanA avoparcin/vancomycin resistance encoding gene cluster. pVEF3 encodes the TA system omega-epsilon-zeta, and plasmid stability tests and transcription analysis show that omega-epsilon-zeta is functional in Enterococcus faecalis OGIX, although with decreasing effect over time. The predicted ABC transporter was not found to confer reduced susceptibility to any of the 28 substances tested. The TA system identified in the pVEF-type plasmids may contribute to vanA plasmid persistence on Norwegian poultry farms. However, size and compositional heterogeneity among E. faecium vanA plasmids suggest that additional plasmid maintenance systems in combination with host specific factors and frequent horizontal gene transfer and rearrangement causes the observed plasmid composition and distribution patterns.

Clinical isolates of Staphylococcus aureus from the Arkhangelsk region, Russia: antimicrobial susceptibility, molecular epidemiology, and distribution of Panton-Valentine leukocidin genes.

A total of 91 consecutive clinical isolates of Staphylococcus aureus were collected at the Regional Hospital of Arkhangelsk, Russia, from May to December 2004, and examined for antimicrobial susceptibility, methicillin resistance and presence of Panton-Valentine leucocidin (PVL) genes. Epidemiological typing was performed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Methicillin-resistant S. aureus (MRSA) isolates were examined by staphylococcal cassette chromosome mec (SCCmec) typing. High-to-moderate rates of resistance to penicillin (beta-lactamase production; 93%), tetracycline (40%), erythromycin and clindamycin (32%) were observed. Forty out of ninety-one (44%) isolates were positive for PVL genes. Thirty-six (40%) PVL-positive methicillin-susceptible S. aureus (MSSA) strains were shown by PFGE and MLST typing (ST121, ST681, ST837) to be part of a nosocomial outbreak caused by clonal complex (CC) 121. PFGE, MLST and SCCmec typing revealed three MRSA clones. Sequence type (ST) 239-III (n=11), ST1097-III (n=1) and ST8-IV (n=3) belong to CC8 of epidemic multiresistant MRSA, whereas ST426-MRSA-IV/CC395 (n=1) has not been reported previously. All MRSA strains were PVL negative. The overall results underline the necessity of microbiological sampling, antimicrobial susceptibility testing, and epidemiological typing as a rational basis for antimicrobial treatment of S. aureus infections, and infection control measures to limit the spread of multiresistant MRSA and epidemic MSSA clones.

VanA-type enterococci from humans, animals, and food: species distribution, population structure, Tn1546 typing and location, and virulence determinants.

VanA-type human (n=69), animal (n=49), and food (n=36) glycopeptide-resistant enterococci (GRE) from different geographic areas were investigated to study their possible reservoirs and transmission routes. Pulsed-field gel electrophoresis (PFGE) revealed two small genetically related clusters, M39 (n=4) and M49 (n=13), representing Enterococcus faecium isolates from animal and human feces and from clinical and fecal human samples. Multilocus sequence typing showed that both belonged to the epidemic lineage of CC17. purK allele analysis of 28 selected isolates revealed that type 1 was prevalent in human strains (8/11) and types 6 and 3 (14/15) were prevalent in poultry (animals and meat). One hundred and five of the 154 VanA GRE isolates, encompassing different species, origins, and PFGE types, were examined for Tn1546 type and location (plasmid or chromosome) and the incidence of virulence determinants. Hybridization of S1- and I-CeuI-digested total DNA revealed a plasmid location in 98% of the isolates. Human intestinal and animal E. faecium isolates bore large (>150 kb) vanA plasmids. Results of PCR-restriction fragment length polymorphism and sequencing showed the presence of prototype Tn1546 in 80% of strains and the G-to-T mutation at position 8234 in three human intestinal and two pork E. faecium isolates. There were no significant associations (P>0.5) between Tn1546 type and GRE source or enterococcal species. Virulence determinants were detected in all reservoirs but were significantly more frequent (P<0.02) among clinical strains. Multiple determinants were found in clinical and meat Enterococcus faecalis isolates. The presence of indistinguishable vanA elements (mostly plasmid borne) and virulence determinants in different species and PFGE-diverse populations in the presence of host-specific purK housekeeping genes suggested that all GRE might be potential reservoirs of resistance determinants and virulence traits transferable to human-adapted clusters.

Comparative DNA analysis of two vanA plasmids from Enterococcus faecium strains isolated from poultry and a poultry farmer in Norway.

The DNA sequences of two plasmids carrying vanA, pVEF1 (39,626 bp) and pVEF2 (39,714 bp), were determined. Forty-three shared coding sequences were identified, and the only nucleotide difference was an 88-bp indel. A postsegregational killing system was identified. This system possibly explains the persistence of the vanA gene cluster in Norwegian poultry farms.

Macrolide-resistant Streptococcus pyogenes in Norway: population structure and resistance determinants.

A 2.7% prevalence of macrolide resistance in 1,657 Norwegian clinical Streptococcus pyogenes isolates was primarily due to erm(TR) (59%) and mef(A) (20%). Four clonal complexes comprised 75% of the strains. Macrolide resistance in S. pyogenes in Norway is imported as resistant strains or locally selected in internationally disseminated susceptible clones.