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Objectives Our objective was to evaluate and exploit a whole genome sequence (WGS) bioinformatics pipeline for predicting antimicrobial resistance and capsular serotypes from invasive group B streptococci (iGBS). Methods For 1975 iGBS recovered during 2015 from CDC's Active Bacterial Core surveillance, we compared pipeline predictions with broth dilution testing. Fifty-six isolates from earlier surveillance were included for testing β-lactams. Conventional serotyping was compared to WGS-based assignments for 302 isolates. Results All 28 isolates with reduced susceptibility to β-lactam antibiotics harboured one of 19 rare PBP2x types. Resistances to erythromycin/clindamycin (808/1975 isolates, 41.0%), erythromycin (235/1975, 11.9%) and lincosamide/streptogramin A/pleuromutilins (56/1975, 2.8%) were predicted by the presence of erm -methylase, mef and lsa determinants, respectively (41 of 56 lsa gene-positive isolates also contained lnu , erm and/or mef genes). Presence of both erm and lsa determinants (25 isolates) predicted non-susceptibility to quinupristin/dalfopristin. Most isolates (1680/1975, 85.1%) were tet gene-positive, although 41/1565 (2.6%) tetM -positive isolates were tetracycline-susceptible. All 53 fluoroquinolone-resistant isolates contained ParC and/or GyrA substitutions. Resistances to rifampin (eight isolates), trimethoprim, chloramphenicol and vancomycin (two isolates each) were predicted by the pipeline. Resistance to macrolides/lincosamides without pipeline prediction was rare and correlated to divergent resistance genes or rRNA A2062G substitution. A selection of 267 isolates assigned WGS-based serotypes were also conventionally serotyped. Of these, 246 (92.1%) were in agreement, with the remaining 21 (7.8%) conventionally non-serotypeable. For 32 of 1975 isolates (1.6%), WGS-based serotypes could not be assigned. Conclusion The WGS-based assignment of iGBS resistance features and serotypes is an accurate substitute for phenotypic testing.

Three invasive Streptococcus pneumoniae strains nonsusceptible to linezolid were isolated in the United States between 2001 and 2012 from the CDC's Active Bacterial Core surveillance. Linezolid binds ribosomal proteins where structural changes within its target site may confer resistance. Our study identified mutations and deletions near the linezolid binding pocket of two of these strains within the rplD gene, which encodes ribosomal protein L4. Mutations in the 23S rRNA alleles or the rplV gene were not detected.

Objectives Multiple invasive group A Streptococcus (GAS) infections were reported to public health by a skilled nursing facility (facility A) in Illinois between May 2014 and August 2016. Cases continued despite interventions including antibiotic prophylaxis for all residents and staff. Two other geographically close facilities reported contemporaneous outbreaks of GAS. We investigated potential reasons for ongoing transmission. Methods We obtained epidemiologic data from chart review of cases and review of facility and public health records from previous investigations into the outbreak. Infection control practices at facility A were observed and evaluated. Whole genome sequencing followed by phylogenetic analysis was performed on available isolates from the three facilities. Results From 2014 to 2016, 19 invasive and 60 noninvasive GAS infections were identified at facility A occurring in three clusters. Infection control evaluations during clusters 2 and 3 identified hand hygiene compliance rates of 14% to 25%, appropriate personal protective equipment use in only 33% of observed instances, and deficient wound-care practices. GAS isolates from residents and staff of all three facilities were subtype emm89.0; on phylogenetic analysis, facility A isolates were monophyletic and distinct. Conclusions Inadequate infection control and improper wound-care practices likely led to this 28-month-long outbreak of severe infections in a skilled nursing facility. Whole genome sequencing and phylogenetic analysis suggested that intrafacility transmission of a single highly transmissible GAS strain was responsible for the outbreak in facility A. Integration of genomic epidemiology tools with traditional epidemiology and infection control assessments was helpful in investigation of a facility-wide outbreak.

In 2009, Streptococcus pneumoniae, or pneumococcus, caused approximately 44 000 cases of invasive pneumococcal disease (IPD) and about 5000 deaths in the United States [1]. In addition, pneumococcus is responsible for a considerable burden of respiratory disease globally [2]. Antibiotic nonsusceptibility is a major concern; understanding the dynamics by which pneumococcus develops nonsusceptibility will help maintain the efficacy of these drugs. There are currently over 90 known serotypes of S. pneumoniae, of which only a small proportion account for most invasive disease and most nonsusceptible infections in the United States. Before introduction of the 7-valent pneumococcal conjugate vaccine (PCV7) in the United States in 2000, the proportion of penicillin-nonsusceptible and macrolide-nonsusceptible isolates from invasive disease was increasing [3–5]. Previous studies have correlated differential use of antimicrobials with different rates of nonsusceptibility across geographic areas, as well as changing patterns of usage and nonsusceptibility over time [6–11]. When PCV7 was introduced, the 7 serotypes included in the vaccine accounted for 78% of penicillin-nonsusceptible strains [3]. In the decade since, rates of disease caused by these 7 serotypes declined by 99%, and serotypes not included in PCV7 have increased significantly, a phenomenon known as serotype replacement. Some of the replacing serotypes (eg, 19A, 15A, 23A, 35B, 6C) have shown high and/or increasing levels of antimicrobial nonsusceptibility, raising the question of whether antimicrobial use may in part be responsible for the increased prevalence [12–14]. In the pre-PCV7 era, geographic differences in proportions of nonsusceptibility to penicillin and macrolides resulted from geographic variation in selective pressure for nonsusceptibility [10, 15]. In pneumococci, selective pressure may affect nonsusceptibility in 2 ways: by increasing the prevalence of nonsusceptibility within each serotype, and/or by increasing the prevalence of serotypes in which the prevalence of nonsusceptibility is high. The frequencies of pneumococcal serotypes within a population reflect multiple selective pressures imposed by natural and vaccine-induced host immunity [16], and it is in principle possible that selection by antimicrobial use may also affect this pattern. In the pre-PCV7 era, no evidence of such an effect was found; the impact of differences in antimicrobial selective pressure was seen solely in varying prevalence of nonsusceptibility within each serotype, but not in variations in the prevalence of the more nonsusceptible serotypes [15]. Understanding the interplay between antimicrobial use pressures and changing serotype distributions has important policy implications for both antimicrobial stewardship and vaccination programs. In this study, we assessed geographical, serotype, and temporal variation in the frequency of drug nonsusceptibility, with a particular focus on the question of whether, contrary to pre-PCV7 observations, there was evidence that geographic variation in the intensity of selective pressure for nonsusceptibility was reflected in greater prevalence of nonsusceptible serotypes.

Background Streptococcus agalactiae (Group B Streptococcus, GBS) is a leading cause of meningitis, sepsis and pneumonia in neonates in the United States. GBS also causes invasive disease in older infants, pregnant women, children and young adults with underlying medical conditions, and older adults. Resistance to lincosamides in the absence of erythromycin resistance is rare in GBS, but has been previously reported in clinical isolates, both on its own or in combination with resistance to streptogramins A and pleuromutilins (L/LSA/LSAP phenotypes). Objectives To retrospectively screen the Active Bacterial Core surveillance (ABCs) GBS isolate collection for these phenotypes in order to identify the causal genetic determinants and determine whether their frequency is increasing. Methods Based on MIC data, 65 (0.31%) isolates susceptible to erythromycin (MIC ≤0.25 mg/L) and non-susceptible to clindamycin (MIC ≥0.5 mg/L) were identified among 21 186 GBS isolates. Genomic DNA was extracted and WGS was performed. The presence of 10 genes previously associated with LSA resistance was investigated by read mapping. Results Forty-nine (75%) isolates carried the lsa (C) gene and expressed the LSAP phenotype, and 12 (18%) carried both the lnu (B) and lsa (E) genes and expressed the LSAP phenotype. The four remaining isolates were negative for all determinants investigated. Conclusions While the overall observed frequency of these phenotypes among our GBS isolates was quite low (0.31%), this frequency has increased in recent years. To the best of our knowledge, this is the first time the LSAP phenotype has been reported among GBS isolates from the USA.

Streptococcus pneumoniae (pneumococcus) remains a major cause of pneumonia, meningitis, and sepsis worldwide, especially in young children. The 7-valent pneumococcal polysaccharide conjugate vaccine, PCV7 (Prevnar (Prevenar); Pfizer), contains capsule polysaccharide antigen of 7 serotypes (4, 6B, 9V, 14, 18C, 19F, and 23F). Its routine use among infants has had a significant impact on vaccine serotype (VT) invasive pneumococcal disease (IPD) and nasopharyngeal (NP) carriage among age groups targeted for vaccination and other age groups; however, concomitant increases in IPD and carriage due to serotypes not included in the vaccine (nonvaccine serotypes [NVTs]) have been observed [1–4]. Pneumococcal NP carriage studies conducted during (1998–2000) and immediately after (2001–2002) a community-randomized PCV7 efficacy trial among Navajo and White Mountain Apache children also identified increased carriage with NVTs [5–7]. The increased prevalence of some vaccine-associated serotypes (VATs, a subgroup of NVTs defined as serotypes within the same serogroup as VTs) and other NVT strains since routine use of PCV7 has been temporally associated with observed increases in the rate of NVT IPD in the United States and in other routine-use countries [8–10]. However, the increased incidence of NVT IPD does not tell us whether NVTs have an increased invasive potential or whether the niche left empty by VTs has only given NVTs more opportunity to colonize and therefore more opportunity to cause disease. Invasive potentials of serogroups in the absence of vaccination have been shown to be geographically and temporally stable [11]. The objective of this study was to determine serotype-specific carriage among Navajo and White Mountain Apache children and adults after 8 years of routine PCV7 use and 12 years after initial use in their communities and to assess whether invasive potentials of serotypes have changed in the setting of long-term PCV7 use. These communities are known to have high prevalence of pneumococcal colonization and IPD rates >4-fold that of the general US population [12]. Based on findings from pre-PCV7 studies [11], we hypothesized that there would be no change in the invasive potential of pneumococcal serotypes. Finally, analysis of individual and household characteristics can identify risk factors associated with carriage in this population. Because IPD episodes are preceded by pneumococcal colonization, identifying modifiable carriage risk factors informs additional disease prevention methods.

The measurement of pneumococcal carriage in the nasopharyngeal reservoir is subject to potential confounders that include low-density and multiple-strain colonization. To compare different methodologies, we picked a random sampling of 100 nasopharyngeal specimens recovered from infants less than 2 years of age who were previously assessed for pneumococcal carriage and serotypes by a conventional method that used direct plating from the transport/storage medium (50 specimens were culture negative and 50 specimens were culture positive for pneumococci). We used a broth enrichment approach and a conventional PCR approach (with and without broth enrichment) to determine pneumococcal carriage and serotypes, and the results were compared to the initial conventional culture-based results. Additionally, we used a lytA -targeted real-time PCR for pneumococcal detection. Broth enrichment for both the culture-based and the PCR-based methods enhanced the isolation of pneumococci and detection of serotype diversity, with the most effective serotype deduction method being one that used broth enrichment prior to sequential multiplex PCR. Similarly, we also found that broth enrichment followed by the lytA -specific real-time PCR was the most sensitive for the detection of apparent pneumococcal carriage. The broth enrichment, conventional multiplex PCR, and real-time PCR approaches used in this study were effective in detecting pneumococcal carriage in the 50 specimens that were negative by conventional direct plating from transport medium (range of numbers of positive specimens, 8/50 to 22/50 [16 to 44%]), and the three different serotyping approaches that used broth enrichment increased the number of serotype identifications from the 100 specimens (12 to 29 additional serotype identifications to be positive). A PCR-based approach that employed a broth enrichment step appeared to best enhance the detection of mixed serotypes and low-density pneumococcal carriage.

Using sequence analysis to detect variation within the hypervariable M protein N terminus, we found 41 emm types encompassing 81 subtypes, among 1064 consecutive invasive group A streptococcus isolates from a recent multistate, population-based surveillance. Seventeen of the 30 emm types represented by multiple isolates displayed multiple subtypes. Most subtypes differed from reference strain emm sequences as a result of single base substitutions or other alterations likely to be stably inherited. The Centers for Disease Control and Prevention database (available at: http://www .cdc.gov/ncidod/biotech/strep/strepblast.htm) currently contains 225 distinct emm types encompassing 450 subtypes. Although this subtyping scheme increases specificity, limited variation within individual types favors introduction of M protein type‐specific vaccines. Group A streptococcus (GAS)–mediated disease is a major problem worldwide. One promising candidate vaccine undergoing clinical trials is a 26-valent vaccine based on the N-terminal type– specific M protein regions that represent common invasive strains in the United States and M types historically associated with rheumatic fever [1]. The surface M protein is thought to be the major virulence factor and major protective antigen of GAS [2]. Protective, opsonic antibodies are elicited by epitopes located within the amino-terminal 50 residues of the M protein [3–5]. For this reason, a precise subtyping scheme based on the 150 bases encoding the mature M protein N terminus appears to be

Pneumococcal macrolide resistance is usually expressed as one of two phenotypes: the M phenotype conferred by the mef gene or the MLSB phenotype caused by modification of ribosomal targets, most commonly mediated by an erm methylase. Target-site modification leading to antibiotic resistance can also occur due to sequence mutations within the 23S rRNA or the L4 and L22 riboproteins. We screened 4,535 invasive isolates resistant to erythromycin and 18 invasive isolates nonsusceptible to quinupristin–dalfopristin (Q-D) to deduce the potential mechanisms involved. Of 4,535 erythromycin-resistant isolates, 66.2% were polymerase chain reaction (PCR)-positive for mef alone, 17.8% for ermB alone, and 15.1% for both mef and ermB. Thirty-seven isolates (0.9%) were PCR negative for both determinants. Of these, 3 were positive for ermA (subclass ermTR) and 25 had chromosomal mutations. No chromosomal mutations (in 23S rRNA, rplD, or rplV) nor any of the macrolides/lincosamides/streptogramin (MLS) resistance genes screened for (ermT, ermA, cfr, lsaC, and vgaA) were found in the remaining nine isolates. Of 18 Q-D nonsusceptible isolates, 14 had chromosomal mutations and one carried both mef and ermB; no chromosomal mutations or other resistance genes were found in 3 isolates. Overall, we found 28 mutations, 13 of which have not been previously described in Streptococcus pneumoniae. The role of these mutations remains to be confirmed by transformation assays.

Background Young children played a major role in pneumococcal nasopharyngeal carriage, acquisition, and transmission in the era before pneumococcal conjugate vaccine (PCV) use. Few studies document pneumococcal household dynamics in the routine-PCV7 era. Methods We investigated age-specific acquisition, household introduction, carriage clearance, and intra-household transmission in a prospective, longitudinal, observational cohort study of pneumococcal nasopharyngeal carriage in 300 American Indian households comprising 1,072 participants between March 2006 and March 2008. Results Pneumococcal acquisition rates were 2–6 times higher in children than adults. More household introductions of new pneumococcal strains were attributable to children

From 1994 through 1999, the available isolates (4148 isolates) from active population-based surveillance of invasive pneumococcal disease in metropolitan Atlanta were serotyped and were tested for antimicrobial susceptibility. Macrolide-resistant isolates were studied for the presence of ermAM (a ribosomal methylase gene), mefE (a macrolide efflux gene), and tetM (the class M tetracycline resistance gene). Macrolide resistance increased from 16% of all invasive isolates in 1994 to 32% in 1999. Of the macrolide-resistant pneumococcal isolates studied, 99% contained genomic copies of mefE or ermAM. Isolates with ermAM were mainly serotypes 6B, 23F, 14, or 19F and contained tetM; mefE-associated isolates were predominantly serotypes 14, 6A, or 19F, and most did not contain tetM. The frequency of the ermAM-mediated phenotype in invasive Streptococcus pneumoniae remained stable over the 6-year surveillance. However, the mefE-mediated phenotype increased from 9% in 1994 to 26% of all isolates in 1999 and was noted in new serotypes. By 1999, 93% of the mefE-containing strains had minimum inhibitory concentrations >/=8 microgram/mL. Dissemination of the mefE determinant accounted for the rapid increase in the rate of macrolide resistance in our S. pneumoniae population.

Detection of group B Streptococcus (GBS) strains at various bacterial concentrations was evaluated using three pigment-producing broth media. At 10 3 CFU/ml, StrepB carrot broth (SBCB), Granada instant liquid biphasic (IGLB), and Northeast Laboratory GBS screening medium (NEL-GBS) showed 100% detection, but at the lower bacterial counts, SBCB and IGLB were more sensitive than NEL-GBS after 24 h.

Eleven isolates representing five distinct outbreaks of pneumococcal conjunctivitis were examined for phenotypic and genetic characteristics. None of the strains possessed capsules, and all strains were susceptible to optochin, bile soluble, and Gen-Probe AccuProbe test positive. All 11 isolates were confirmed as Streptococcus pneumoniae by DNA-DNA reassociation experiments.

Background. A second-generation 13-valent pneumococcal conjugate vaccine, PCV13, was recently licensed. Although PCV13 includes serotype 6A, the usefulness of that antigen may be limited by the emergence of a new serotype, 6C, which was identified among isolates initially characterized (Quellung reaction) as serotype 6A. The epidemiology of serotype 6C prior to and after 7-valent PCV (PCV7) introduction is incompletely understood. Methods. We analyzed conventionally serotyped 6A (CS6A) pneumococci from invasive disease case patients of all ages and carriage isolates from children and adults obtained in population-based studies among Navajo and White Mountain Apache communities during 1994-2009. Samples were tested by triplex polymerase chain reaction to resolve serotypes 6C and 6A. Results. A total of 74 invasive CS6A episodes occurred. All were retyped by polymerase chain reaction; 40 (54.1%) were serotype 6C. The mean annual incidence of serotype 6C invasive disease was 0.3 (95% confidence interval, 0.03-0.9), 0.7 (95% confidence interval, 0.2-1.3), and 1.5 (95% confidence interval, 1.0-2.1) cases per 100,000 population in the years prior to the PCV7 efficacy trial, during the time the PCV7 trial was conducted, and following PCV7 introduction and routine use, respectively (P = .01). In the routine vaccination era, 76% of invasive CS6As were serotype 6C; nearly all cases occurred in adults. The proportion of serotype 6C among CS6A carriage isolates increased from 42% to 61% to 94% in the prevaccine, early vaccine, and routine vaccination eras, respectively. Conclusion. In the PCV7 routine use era, virtually all serogroup 6 invasive pneumococcal disease and carriage strains among Navajo and White Mountain Apache communities are 6C. Monitoring and evaluation of this and other emerging serotypes among invasive disease and carriage isolates is warranted.

Background. Group B Streptococcus (GBS), traditionally considered to be a neonatal pathogen, is an important cause of morbidity and mortality among older adults and among those with underlying medical conditions. We used population-based surveillance to examine trends in adult GBS disease during the period 1990-2007 and to describe the epidemiology of adult GBS disease to guide prevention efforts. Methods. Active Bacterial Core surveillance was conducted in selected counties in 10 US states. A case was defined as isolation of GBS from a normally sterile site in a nonpregnant resident of a surveillance area who was ≥ 18 years of age. Rates were calculated using US Census data. Demographic and clinical information was abstracted from medical records. Serotyping and susceptibility testing were performed on isolates collected from a subset of case patients. Results. A total of 19,512 GBS cases were identified in nonpregnant adults during 1990-2007 (median patient age, 63 years); the incidence of adult GBS disease doubled from 3.6 cases per 100,000 persons during 1990 to 7.3 cases per 100,000 persons during 2007 (P

Background. Widespread use of pneumococcal conjugate vaccine (PCV7) resulted in decreases in invasive disease among children and elderly persons. The benefits may be offset by increases in disease due to serotypes not included in the vaccine (hereafter, "nonvaccine serotypes"). We evaluated the effect of PCV7 on incidence of disease due to nonvaccine serotypes. Methods. Cases of invasive disease were identified in 8 geographic areas through the Centers for Disease Control and Prevention's Active Bacterial Core surveillance. Serotyping and susceptibility testing of isolates were performed. We calculated the incidence of disease for children aged

BACKGROUND: The introduction of conjugate pneumococcal vaccination for children has reduced the burden of invasive disease due to pneumococcal conjugate vaccine (PCV) types (i.e., serotypes 9V, 14, 6B, 18C, 23F, 19F, and 4) in adults. As nonvaccine serotypes become predominant causes of invasive disease among adults, it is necessary to evaluate the disease severity and mortality associated with infection due to nonvaccine serotypes, compared with PCV serotypes, in adults. METHODS: The association of pneumococcal serotype and host-related variables with disease severity and mortality was statistically examined (with multivariable analysis) in 796 prospectively enrolled, hospitalized adult patients with bacteremia due to Streptococcus pneumoniae. RESULTS: In multivariate analyses of risk in patients with invasive pneumococcal disease, older age (age, > or = 65 years; P = .004), underlying chronic disease (P = .025), immunosuppression (P = .035), and severity of disease (P < .001) were significantly associated with mortality; no association was found between nosocomial infection with invasive serotypes 1, 5, and 7 and mortality. The risk factors meningitis (P = .001), suppurative lung complications (P < or = .001), and preexisting lung disease (P = .051) were significantly associated with disease severity, independent of infecting serotype. No differences were seen in disease severity or associated mortality among patients infected with PCV serotypes, compared with patients infected with nonvaccine serotypes. CONCLUSIONS: Our data support the notion that host factors are more important than isolate serotype in determining the severity and outcome of invasive pneumococcal disease and that these outcomes are unlikely to change in association with nonvaccine serotype infection in the post-conjugate vaccine era.

We have identified an unusual group of viridans group streptococci that resemble Streptococcus pneumoniae . DNA-DNA homology studies suggested that a subset of these isolates represent a novel species that may be included in the S. oralis- S. mitis group of viridans group streptococci. We suggest that this novel species be termed Streptococcus pseudopneumoniae . A combination of phenotypic and genetic reactions allows its identification. S. pseudopneumoniae strains do not have pneumococcal capsules, are resistant to optochin (inhibition zones, less than 14 mm) when they are incubated under an atmosphere of increased CO 2 but are susceptible to optochin (inhibition zones, >14 mm) when they are incubated in ambient atmospheres, are not soluble in bile, and are positive by the GenProbe AccuProbe Pneumococcus test. The bile solubility test is more specific than the optochin test for identification of S. pneumoniae . Genetic tests for pneumolysin ( ply ) and manganese-dependent superoxide dismutase ( sodA ) and identification tests with a commercial probe, AccuProbe Pneumococcus, do not discriminate between the new species and S. pneumoniae .

Restriction digest profiling of pneumococcal pbp2b -specific amplicons was effective for screening penicillin resistance. The pbp2b amplicon of all pneumococcal isolates for which the MICs of penicillin were ≤0.03 μg/ml had one of two different susceptible restriction profiles, and all 33 isolates for which MICs were 0.5 μg/ml or greater had one of seven distinct resistant profiles. Low-concentration penicillin resistance (MICs = 0.06 μg/ml to 0.25 μg/ml) was associated with sensitive Hae III profiles in some isolates; however, Rsa I profiling and pbp2b sequence analysis of such isolates revealed that some isolates contained low-level resistant pbp2b alleles, while others had susceptible pbp2b alleles. This data indicates that low-level penicillin resistance is sometimes conferred by determinants other than pbp2b .