Erythromycin-nonsusceptible Streptococcus pneumoniae in Children, 1999–2001

After increasing from 1995 to 1999, invasive erythromycin-nonsusceptible Streptococcus pneumoniae rates per 100,000 decreased 53.6% in children from Baltimore, Maryland (US), from 1999 to 2001, which was partially attributed to strains related to the mefE-carrying England14-9 clone. The decline in infection rates was likely due to the pneumococcal 7-valent conjugate vaccine

Monitoring the Long-Term Molecular Epidemiology of the Pneumococcus and Detection of Potential ‘Vaccine Escape’ Strains

While the pneumococcal protein conjugate vaccines reduce the incidence in invasive pneumococcal disease (IPD), serotype replacement remains a major concern. Thus, serotype-independent protection with vaccines targeting virulence genes, such as PspA, have been pursued. PspA is comprised of diverse clades that arose through recombination. Therefore, multi-locus sequence typing (MLST)-defined clones could conceivably include strains from multiple PspA clades. As a result, a method is needed which can both monitor the long-term epidemiology of the pneumococcus among a large number of isolates, and analyze vaccine-candidate genes, such as pspA, for mutations and recombination events that could result in 'vaccine escape' strains.We developed a resequencing array consisting of five conserved and six variable genes to characterize 72 pneumococcal strains. The phylogenetic analysis of the 11 concatenated genes was performed with the MrBayes program, the single nucleotide polymorphism (SNP) analysis with the DNA Sequence Polymorphism program (DnaSP), and the recombination event analysis with the recombination detection package (RDP).The phylogenetic analysis correlated with MLST, and identified clonal strains with unique PspA clades. The DnaSP analysis correlated with the serotype-specific diversity detected using MLST. Serotypes associated with more than one ST complex had a larger degree of sequence polymorphism than a serotype associated with one ST complex. The RDP analysis confirmed the high frequency of recombination events in the pspA gene.The phylogenetic tree correlated with MLST, and detected multiple PspA clades among clonal strains. The genetic diversity of the strains and the frequency of recombination events in the mosaic gene, pspA were accurately assessed using the DnaSP and RDP programs, respectively. These data provide proof-of-concept that resequencing arrays could play an important role within research and clinical laboratories in both monitoring the molecular epidemiology of the pneumococcus and detecting 'vaccine escape' strains among vaccine-candidate genes

Characterization of In Vitro Biofilm-Associated Pneumococcal Phase Variants of a Clinically Relevant Serotype 3 Clone

An increasing proportion of children with acute otitis media due to Streptococcus pneumoniae have serotype 3 infections since licensure of the seven-valent pneumococcal conjugate vaccine. These serotype 3 strains are genetically related by molecular subtyping. During otitis media with effusion and recurrent otitis media, biofilms commonly develop. Pneumococcal in vitro biofilms are comprised of phase variants that differ in colony morphology. By using a representative strain of the mucoid serotype 3 clone, rough phase variants with a diverse array of mutations were detected in biofilms formed in vitro. Most phase variants had mutations in the cps3D gene, the first gene of the capsular operon. Eleven had single nucleotide polymorphisms (SNPs) in the cps3D gene, one had an SNP in the −10 promoter, and three had large deletions in the cps3D gene. Reversion to the mucoid phenotype was associated with reversion of the mutation in the cps3D gene. Unlike the phase variants detected in the nasopharynx, which have at least 20% of the parental amount of capsule, the in vitro biofilm-associated phase variants had ≤12% of the parental amount of capsule, as determined by capsule enzyme-linked immunosorbent assays. Using real-time reverse transcription-PCR, we determined that capsule expression in the phase variants was likely regulated at multiple levels. These in vitro phase variation data, which underscore the plasticity of the pneumococcus, need to be confirmed with in vivo analyses of the middle ear mucosa during otitis media

Locus-Specific Mutational Events in a Multilocus Variable-Number Tandem Repeat Analysis of Escherichia coli O157:H7

Multilocus variable-number tandem repeat analysis (MLVA) is a validated molecular subtyping method for detecting and evaluating Escherichia coli O157:H7 outbreaks. In a previous study, five outbreaks with a total of 21 isolates were examined by MLVA. Nearly 20% of the epidemiologically linked strains were single-locus variants (SLV) of their respective predominant outbreak clone. This result prompted an investigation into the mutation rates of the seven MLVA loci (TR1 to TR7). With an outbreak strain that was an SLV at the TR1 locus of the predominant clone, parallel and serial batch culture experiments were performed. In a parallel experiment, none (0/384) of the strains analyzed had mutations at the seven MLVA loci. In contrast, in the two 5-day serial experiments, 4.3% (41/960) of the strains analyzed had a significant variation in at least one of these loci (P < 0.001). The TR2 locus accounted for 85.3% (35/41) of the mutations, with an average mutation rate of 3.5 × 10(−3); the mutations rates for TR1 and TR5 were 10-fold lower. Single additions accounted for 77.1% (27/35) of the mutation events in TR2 and all (6/6) of the additions in TR1 and TR5. The remaining four loci had no slippage events detected. The mutation rates were locus specific and may impact the interpretation of MLVA data for epidemiologic investigations

Serotype 14 Variants of the France 9V(−3) Clone from Baltimore, Maryland, Can Be Differentiated by the cpsB Gene

European serotype 14 variants of the France 9V(−3) clone, which have arisen through recombination events involving the penicillin binding protein 1a (pbp1a) gene, have cpsB sequences distinct from those of the 9V(−3) clone. Serotype 14 variants of the 9V(−3) clone have not been compared to genetically diverse serotype 14 strains isolated from an entire metropolitan area in the United States. All serotype 14 non-penicillin-susceptible Streptococcus pneumoniae strains causing invasive disease in Baltimore, Md., from 1995 to 1996 were compared by using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), pbp1a PCR restriction profiles, and cpsB and pbp1a sequences. The cpsB genes from strains of 13 serotypes also were analyzed to assess the correlation with serotype. Twenty-seven percent (3 of 11) of the serotype 14 strains were related by PFGE and MLST to the 9V(−3) clone. The serotype 14 variants from Baltimore, unlike the European variants, were related neither to the 9V(−3) clone nor to the R6 strain from positions 1498 to 1710 of the pbp1a gene. All serotype 14 strains had cpsB sequences that differed by ≤1% (0 to 5 of 476 bp) from each other and that were ≥16% (78 to 83 of 476 bp) divergent from that of the 9V(−3) clone. Allowing for a 2-bp difference in the cpsB sequence resulted in the highest correlation between the cpsB gene and serotype. Overall, 95% (84 of 88) of the strains were classified correctly by serotype with the cpsB sequence. The distal recombination site of the Baltimore serotype 14 variants of the 9V(−3) clone was not identical to that of the European serotype 14 variants. The cpsB gene was serotype specific regardless of whether capsular switching occurred. Although the correlation between serotype and the cpsB sequence was high, the overall diversity of the cpsB gene within a serotype likely will limit the role of this gene in a sequence-based serotyping method

Multilocus Variable-Number Tandem Repeat Analysis Distinguishes Outbreak and Sporadic Escherichia coli O157:H7 Isolates

Escherichia coli O157:H7 is a major cause of food-borne illness in the United States. Outbreak detection involves traditional epidemiological methods and routine molecular subtyping by pulsed-field gel electrophoresis (PFGE). PFGE is labor-intensive, and the results are difficult to analyze and not easily transferable between laboratories. Multilocus variable-number tandem repeat (VNTR) analysis (MLVA) is a fast, portable method that analyzes multiple VNTR loci, which are areas of the bacterial genome that evolve quickly. Eighty isolates, including 21 isolates from five epidemiologically well-characterized outbreaks from Pennsylvania and Minnesota, were analyzed by PFGE and MLVA. Strains in PFGE clusters were defined as strains that differed by less than or equal to one band by using XbaI and the confirmatory enzyme SpeI. MLVA was performed by comparing the number of tandem repeats at seven loci. From 6 to 30 alleles were found at the seven loci, resulting in 64 MLVA types among the 80 isolates. MLVA correctly identified the isolates from all five outbreaks if only a single-locus variant was allowed. MLVA differentiated strains with unique PFGE types. Additionally, MLVA discriminated strains within PFGE-defined clusters that were not known to be part of an outbreak. In addition to being a simple and validated method for E. coli O157:H7 outbreak detection, MLVA appears to have a sensitivity equal to that of PFGE and a specificity superior to that of PFGE

SARS-CoV-2 is associated with high viral loads in asymptomatic and recently symptomatic healthcare workers.

BackgroundHealthcare workers (HCW) are at increased risk of SARS-CoV-2 infection from both patients and other HCW with coronavirus disease 2019 (COVID-19). RT-PCR cycle threshold (Ct) values of SARS-CoV-2 ≤ 34 and the first 7-9 days of symptoms are associated with enhanced infectivity. We determined Ct values and duration of symptoms of HCW with a positive SARS-CoV-2 test. As HCW often assume their greatest risk of acquiring SARS-CoV-2 is working on a COVID-19 unit, we also determined Ct values and symptom duration of inpatients with a positive SARS-CoV-2 test.MethodsFrom 6/24/2020-8/23/2020, Ct values and duration of symptoms from 13 HCW, 12 outpatients, and 28 inpatients who had a positive nasopharyngeal swab for SARS-CoV-2 were analyzed.ResultsAmong HCW with a positive SARS-CoV-2 test, 46.2% (6/13) were asymptomatic and requested testing due to an exposure to someone with COVID-19; 83.3% (5/6) of those exposures occurred in the community rather than in the hospital. The median Ct value of HCW was 23.2, and 84.6% (11/13) had a Ct value ≤ 34. The median Ct value of 29.0 among outpatients with COVID-19 did not significantly differ from HCW. In contrast, inpatients with a positive SARS-CoV-2 test had a median Ct value of 34.0 (p = 0.003), which translated into a median ~1,000-fold lower viral load than observed in HCW. Among those with symptoms related to COVID-19, no (0/6) HCW compared to 50% (6/12) of inpatients had symptoms for at least one week (p = 0.04).ConclusionsAt our institution, asymptomatic COVID-19 accounted for nearly half of the cases among HCW. Symptomatic HCW had high viral loads and short duration of symptoms, both of which are associated with peak infectivity. Infection prevention programs should educate HCW on these findings in an effort to increase adherence to the requirement to maintain six feet separation in workspaces and breakrooms, in addition to consistently wearing personal protection equipment