BACKGROUND:The increase in carbapenem-resistant Enterobacteriaceae (CRE) infections is a critical public health issue. We recently experienced the largest single-center pediatric outbreak of carbapenem-resistant Klebsiella pneumoniae (CRKP) at our hospital. The objective of this study was to describe the molecular epidemiology of this outbreak before and after infection-prevention interventions. METHODS:All positive cultures and associated clinical conditions were reviewed to determine whether health care-associated infections (HAIs) exist. HAIs were defined using Centers for Disease Control and Prevention guidelines. CRKP isolates were collected and screened for the presence of β-lactamase genes. Strain relatedness of CRKP isolates was determined by field-inversion gel electrophoresis (FIGE) and multilocus sequence typing (MLST). Polymerase chain reaction (PCR) amplification and sequencing of blaTEM, blaSHV, and blaKPC genes were performed on representative isolates. RESULTS:During March-July 2010, 18 CRKP isolates were recovered from 15 unique patients. Six isolates were considered HAIs; all were central-line-associated bloodstream infections. All isolates testing positive by PCR for blaKPC were found to carry KPC-3 in transposon Tn4401 isotype "b." FIGE revealed 2 prevalent patterns (accounting for 10 and 3 CRKP isolates, respectively) that MLST demonstrated to consist entirely of strains from ST730; the remaining FIGE types corresponded to ST14, ST15, and ST1559 (a single-locus variant of ST730), with these alternate backgrounds appearing later in the outbreak. New CRKP cases decreased after the implementation of infection-control interventions. All isolates were ciprofloxacin sensitive. CONCLUSIONS:Molecular analyses document the introduction of a KPC-3-producing CRKP clone into our hospital setting, though some isolates appear to have other mechanisms of carbapenem resistance. The transition to a polyclonal epidemiology suggests that the initial outbreak was due to nosocomial spread of a single ST730 clone, while latter isolates may have been secondary to the introduction of a blaKPC-3/Tn4401 isotype "b"-containing plasmid into other K pneumoniae strain backgrounds versus new carbapenemase-producing bacteria.

Background: Surveillance of children with acute otitis media (AOM) for nasopharyngeal colonization with Streptococcus pneumoniae before, during and after the introduction of 7-valent pneumococcal conjugate vaccine (PCV7) indicated the near-complete elimination of PCV7 strains and the emergence of pneumococcal serotype 19A.Methods: To determine effects of the introduction of 13-valent pneumococcal conjugate vaccine (PCV13) on pneumococcal nasopharyngeal colonization, we obtained nasopharyngeal cultures from 228 children 6 through 23 of age months presenting with a new episode of AOM during 2012 and 2013 and enrolled in an ongoing clinical trial of antimicrobial efficacy. All children had received at least 2 doses of PCV13. The S. pneumoniae isolates were subjected to serotyping and testing for antimicrobial susceptibility. We compared the findings with results obtained in 3 earlier studies.Results: We found nasopharyngeal colonization with S. pneumoniae in 113 (50%) of the children with AOM. PCV7 and PCV13 serotypes accounted for 2% and 12%, respectively, of the pneumococcal isolates. Of the 14 PCV13 isolates, 8 were serotype 19A. Nonvaccine serotypes accounted for 69% of the isolates. Most frequently occurring were sub-types of serotype 15 (23%) and serotype 35B (9%). Overall, 33% of the isolates were penicillin nonsusceptible, a proportion not significantly different from proportions found in our 3 earlier studies (26%, 36% and 37%, respectively). Serotypes 15 and 35B accounted for 51% of penicillin-nonsusceptible isolates.Conclusions: Expansion of contents of pneumococcal vaccine administered to children is followed by not-fully-predictable changes in nasopharyngeal pneumococcal colonization. Continued surveillance is required to help inform future vaccine development.

Children 6 through 23 Months of Age at the Time of Diagnosis of an Episode of Acute Otitis Media (1999-2014) Judith M. Martin, MD; Alejandro Hoberman, MD; Timothy Shope, MD, MPH; Karen A. Barbadora, BS, MT-ASCP; Marcia Kurs-Lasky, MS; Michael Green, MD, MPH; Department of Pediatrics, Children’s Hospital of Pittsburgh, Pittsburgh, PA; Department of Pediatrics, Division of Infectious Disease, Children’s Hospital of Pittsburgh, Pittsburgh, PA

The diagnosis of acute bacterial sinusitis can be challenging because symptoms of acute sinusitis and an upper respiratory tract infection (URI) overlap. A rapid test, if accurate in differentiating sinusitis from URI, could be helpful in the diagnostic process. We examined the utility of nasopharyngeal cultures in identifying the subgroup of children with a clinical diagnosis of acute sinusitis who are least likely to benefit from antimicrobial therapy (those with completely normal sinus radiographs). Nasopharyngeal swabs were collected from 204 children meeting a priori clinical criteria for acute sinusitis. All children had sinus X-rays at the time of diagnosis. To determine if negative nasopharyngeal culture results could reliably identify the subgroup of children with normal radiographs, we calculated negative predictive values and negative likelihood ratios. Absence of pathogens in the nasopharynx was not helpful in identifying this low-risk subgroup.

BACKGROUND Recommendations vary regarding immediate antimicrobial treatment versus watchful waiting for children younger than 2 years of age with acute otitis media. METHODS We randomly assigned 291 children 6 to 23 months of age, with acute otitis media diagnosed with the use of stringent criteria, to receive amoxicillin-clavulanate or placebo for 10 days. We measured symptomatic response and rates of clinical failure. RESULTS Among the children who received amoxicillin-clavulanate, 35% had initial resolution of symptoms by day 2, 61% by day 4, and 80% by day 7; among children who received placebo, 28% had initial resolution of symptoms by day 2, 54% by day 4, and 74% by day 7 (P=0.14 for the overall comparison). For sustained resolution of symptoms, the corresponding values were 20%, 41%, and 67% with amoxicillin-clavulanate, as compared with 14%, 36%, and 53% with placebo (P=0.04 for the overall comparison). Mean symptom scores over the first 7 days were lower for the children treated with amoxicillin-clavulanate than for those who received placebo (P=0.02). The rate of clinical failure--defined as the persistence of signs of acute infection on otoscopic examination--was also lower among the children treated with amoxicillin-clavulanate than among those who received placebo: 4% versus 23% at or before the visit on day 4 or 5 (P<0.001) and 16% versus 51% at or before the visit on day 10 to 12 (P<0.001). Mastoiditis developed in one child who received placebo. Diarrhea and diaper-area dermatitis were more common among children who received amoxicillin-clavulanate. There were no significant changes in either group in the rates of nasopharyngeal colonization with nonsusceptible Streptococcus pneumoniae. CONCLUSIONS Among children 6 to 23 months of age with acute otitis media, treatment with amoxicillin-clavulanate for 10 days tended to reduce the time to resolution of symptoms and reduced the overall symptom burden and the rate of persistent signs of acute infection on otoscopic examination. (Funded by the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT00377260.).

Although there is tremendous interest in understanding the evolutionary roles of horizontal gene transfer (HGT) processes that occur during chronic polyclonal infections, to date there have been few studies that directly address this topic. We have characterized multiple HGT events that most likely occurred during polyclonal infection among nasopharyngeal strains of Streptococcus pneumoniae recovered from a child suffering from chronic upper respiratory and middle-ear infections. Whole genome sequencing and comparative genomics were performed on six isolates collected during symptomatic episodes over a period of seven months. From these comparisons we determined that five of the isolates were genetically highly similar and likely represented a dominant lineage. We analyzed all genic and allelic differences among all six isolates and found that all differences tended to occur within contiguous genomic blocks, suggestive of strain evolution by homologous recombination. From these analyses we identified three strains (two of which were recovered on two different occasions) that appear to have been derived sequentially, one from the next, each by multiple recombination events. We also identified a fourth strain that contains many of the genomic segments that differentiate the three highly related strains from one another, and have hypothesized that this fourth strain may have served as a donor multiple times in the evolution of the dominant strain line. The variations among the parent, daughter, and grand-daughter recombinant strains collectively cover greater than seven percent of the genome and are grouped into 23 chromosomal clusters. While capturing in vivo HGT, these data support the distributed genome hypothesis and suggest that a single competence event in pneumococci can result in the replacement of DNA at multiple non-adjacent loci.

Methods: Before and after introduction of pneumococcal conjugate vaccine (PCV7), the authors obtained nasopharyngeal (NP) specimens from 3 groups of children aged 6 to 23 months with acute otitis media (AOM): group 1 (pre-PCV7), group 2 (early post-PCV7), and group 3 (late post-PCV7). Results: Of the Streptococcus pneumoniae isolates, the proportion that were vaccine serotypes (VTs) declined progressively (60.4% vs 48.6% vs 5.2% in groups 1, 2, and 3, respectively; P < .001). Concurrently, increases occurred in the proportion of penicillin-nonsusceptible isolates (minimum inhibitory concentration >0.1 µg/mL; 26.7% vs 37.8% vs. 38.5%; P = .12); the proportion of isolates that were serotype 19A (4.0% vs 0% vs 25.9%; P < .001); and the proportion of 19A isolates that were penicillin-nonsusceptible (0% in group 1, 68.6% in group 3; P = .004). Conclusion: Shifts in pneumococcal serotype distribution and increases in penicillin nonsusceptibility among pneumococcal isolates from children with AOM underscore the need for continuing bacteriological surveillance for future vaccine development.

BACKGROUND:Streptococcus pneumoniae [Sp] infection is associated with local and systemic disease. Our current understanding of the differential contributions of genetic strain variation, serotype, and host response to disease phenotype is incomplete. Using the chinchilla model of otitis media [OM] we investigated the disease phenotype generated by the laboratory strain TIGR4 and each of thirteen clinical strains (BS68-75, BS290, BS291, BS293, BS436 and BS437); eleven of the thirteen strains have been genomically sequenced.METHODOLOGY/PRINCIPAL FINDINGS:For each strain 100 colony forming units were injected bilaterally into the tympanic bullae of 6 young adult chinchillas under general anesthesia. All animals were examined daily for local and systemic disease by a blinded observer. Pneumatic otoscopy was used to evaluate local disease, and behavioral assessments served as the measure of systemic disease. Virulence scoring was performed using a 4-point scale to assess four clinical parameters [severity and rapidity of local disease onset; and severity and rapidity of systemic disease onset] during a 10-day evaluation period. Highly significant variation was observed among the strains in their ability to cause disease and moribundity.CONCLUSIONS/SIGNIFICANCE:As expected, there was a significant correlation between the rapidity of systemic disease onset and severity of systemic disease; however, there was little correlation between the severity of otoscopic changes and severity of systemic disease. Importantly, it was observed that different strains of the same serotype produced as broad an array of disease phenotypes as did strains of different serotypes. We attribute these phenotypic differences among the strains to the high degree of genomic plasticity that we have previously documented.

MATERIALS AND METHODSDNA sequencing. We obtained eight clinical S. pneumoniae isolates, with different serotypes and multilocus sequence typing (MLST) types, from pediatric patients participating in a Fluzone vaccine trial at the Children’s Hospital of Pittsburgh. The genomes of these strains were sequenced at the Center for Genomic Sciences (CGS) using a 454 Life Sciences GS-20 sequencer (26). Strains were sequenced to a depth of 16-fold or greater and were assembled de novo by the 454 Newbler de novo assembler to 281 contigs per genome, on average. Lander-Waterman statistics predict that more than 99.9% of each genome was sequenced. Regions of repetitive sequence caused most of the assembly gaps. Informal comparison between high-quality Sanger reads and 454 data suggest an error rate of less than 1 in 1,000 bases in each assembly. Most base call errors are single-base insertions or deletions in homo-nucleotide repeats, which can result in artifactual frameshifts. Sequence data for the other nine strains examined came from various sources. Four isolates were sequenced by the S. pneumoniae Sequencing Group at the Sanger Institute: 23F, INV104B, INV200, and OXC141. The sequences of the latter three strains are incomplete and can be obtained from ftp://ftp. sanger. ac. uk/pub/pathogens/spn/. The published genome sequence of strain R6 (GenBank accession number AE007317) was produced by Eli Lilly & Co (19). The Institute for Genomic Research (TIGR) sequenced strains TIGR4 (GenBank accession number AE005672), TIGR670-6B (project information available at http://www. ncbi. nlm. nih. gov …

The distributed-genome hypothesis (DGH) states that pathogenic bacteria possess a supragenome that is much larger than the genome of any single bacterium and that these pathogens utilize genetic recombination and a large, noncore set of genes as a means of diversity generation. We sequenced the genomes of eight nasopharyngeal strains of Streptococcus pneumoniae isolated from pediatric patients with upper respiratory symptoms and performed quantitative genomic analyses among these and nine publicly available pneumococcal strains. Coding sequences from all strains were grouped into 3,170 orthologous gene clusters, of which 1,454 (46%) were conserved among all 17 strains. The majority of the gene clusters, 1,716 (54%), were not found in all strains. Genic differences per strain pair ranged from 35 to 629 orthologous clusters, with each strain's genome containing between 21 and 32% noncore genes. The distribution of the orthologous clusters per genome for the 17 strains was entered into the finite-supragenome model, which predicted that (i) the S. pneumoniae supragenome contains more than 5,000 orthologous clusters and (ii) 99% of the orthologous clusters ( approximately 3,000) that are represented in the S. pneumoniae population at frequencies of >or=0.1 can be identified if 33 representative genomes are sequenced. These extensive genic diversity data support the DGH and provide a basis for understanding the great differences in clinical phenotype associated with various pneumococcal strains. When these findings are taken together with previous studies that demonstrated the presence of a supragenome for Streptococcus agalactiae and Haemophilus influenzae, it appears that the possession of a distributed genome is a common host interaction strategy.

OBJECTIVES:Rates of macrolide resistance in group A streptococci (GAS) were reported to be low in the US in the 1990s. However, we documented an unexpectedly high rate of macrolide resistance among GAS in Pittsburgh, PA, in 2001 and 2002. In an effort to define the current prevalence of macrolide-resistant GAS in the US, a multicentre surveillance project was initiated. METHODS:Between October 2002 and May 2003, 50 pharyngeal GAS isolates per month were requested from each of the nine participating sites representing a wide geographical distribution. Standard susceptibility testing was performed and the macrolide resistance phenotype was assessed using double-disc diffusion testing. Monthly and annual rates of macrolide resistance were calculated for each site. An adjusted overall rate of macrolide resistance was determined to account for differences in the numbers of GAS isolates sent from each centre. RESULTS:Overall, 171 of the 2797 collected isolates of GAS (6.1%) were resistant to erythromycin. The adjusted overall resistance rate was 5.2%. Rates of macrolide resistance varied by site (range 3.0-8.7%) and also by month (<2% to >10%). The M phenotype of macrolide resistance accounted for >60% of all macrolide-resistant isolates recovered in this study. CONCLUSIONS:These data suggest an increasing prevalence and broad geographical distribution of macrolide-resistant GAS in the US, indicating the need for ongoing local and national longitudinal surveillance to define the extent of this problem.

Objective: To evaluate the occurrence of cases of acute rheumatic fever (ARF) in western Pennsylvania although there has been a marked reduction of cases of ARF in the United States overall.Study design: From 1994 to 2003, the cases of ARF evaluated at Children's Hospital of Pittsburgh were reviewed. In addition, throat cultures were performed on a subset of these children and their family members beginning in 1995. Molecular typing was performed on isolates of the group A streptococcus (GAS) recovered, using field inversion gel electrophoresis. ping. (FIGE) and emm typing.Results: There were 121 new cases of ARF from 1994 to 2003. Of these, 57% were male. The median age was 10 years. The majority of children (57%) had carditis with or without another manifestation of ARF. The results of throat cultures were available for 231 persons; 36% (30/84) of the children with ARF and 14% (20/147) of family members were positive for GAS. Eight emm types were observed (emm, 1, 2, 6, 12, 18, 28, 75, and 89). Data suggest that emm type 12 may be a rheumatogenic strain.Conclusion: ARF remains a problem in western Pennsylvania. Identification of emm types associated with cases should enlighten vaccine development.

ABSTRACT Eight low-passage-number Streptococcus pneumoniae clinical isolates, each of a different serotype and a different multilocus sequence type, were obtained from pediatric participants in a pneumococcal vaccine trial. Comparative genomic analyses were performed with these strains and two S. pneumoniae reference strains. Individual genomic libraries were constructed for each of the eight clinical isolates, with an average insert size of ∼1 kb. A total of 73,728 clones were picked for arraying, providing more than four times genomic coverage per strain. A subset of 4,793 clones were sequenced, for which homology searches revealed that 750 (15.6%) of the sequences were unique with respect to the TIGR4 reference genome and 263 (5.5%) clones were unrelated to any available streptococcal sequence. Hypothetical translations of the open reading frames identified within these novel sequences showed homologies to a variety of proteins, including bacterial virulence factors not previously identified in S. pneumoniae . The distribution and expression patterns of 58 of these novel sequences among the eight clinical isolates were analyzed by PCR- and reverse transcriptase PCR-based analyses, respectively. These unique sequences were nonuniformly distributed among the eight isolates, and transcription of these genes in planktonic cultures was detected in 81% (172/212) of their genic occurrences. All 58 novel sequences were transcribed in one or more of the clinical strains, suggesting that they all correspond to functional genes. Sixty-five percent (38/58) of these sequences were found in 50% or less of the clinical strains, indicating a significant degree of genomic plasticity among natural isolates.

Objective. A 4-year longitudinal study of school-aged children was conducted to describe the clinical characteristics and epidemiologic features of infections with group A streptococci (GAS). Methods. Between 1998 and 2002, surveillance throat cultures were performed twice per month (October to May) for a cohort of elementary school children in Pittsburgh, Pennsylvania. In addition, throat cultures were obtained during any respiratory illness. Erythromycin and clindamycin susceptibility testing was performed for all isolates. Molecular typing was performed with field-inversion gel electrophoresis. Representative isolates from each field-inversion gel electrophoresis group were emm typed. Strict definitions were used to characterize each GAS infection. Children were classified into 4 categories each year, ie, single episode, recurrent episodes, carriers of GAS, and no infections. Results. A total of 48 to 100 children per year were studied for 4 years; 61 (49%) were male. The mean age was 9.6 years (range: 5–15 years). A total of 5658 throat cultures were performed; 878 (15.5%) were positive for GAS. Antimicrobial agents were used to treat 209 episodes of infection. Thirteen emm types were observed during the 4-year period. GAS were isolated most often from children who were carriers; isolates from single episodes were next most common. Children carried a single emm type for a mean of 10.8 weeks (range: 3–34 weeks). Carriers were likely to be classified again as carriers in subsequent years and frequently switched emm types. Sixty-two percent of the children had ≥1 year with no infections. Conclusions. GAS infections are common among school-aged children. The majority of positive throat cultures observed in this longitudinal study were obtained from children who were carriers of GAS. Carriers switched emm types but tended to become carriers repeatedly during the study. Practitioners should consider treating children known to be GAS carriers when they develop a new illness that is consistent with streptococcal pharyngitis, because they may acquire new emm types and be at risk for rheumatic heart disease.