Emergence of ciprofloxacin-resistant Neisseria meningitidis in North America.

We report on three cases of meningococcal disease caused by ciprofloxacin-resistant Neisseria meningitidis, one in North Dakota and two in Minnesota. The cases were caused by the same serogroup B strain. To assess local carriage of resistant N. meningitidis, we conducted a pharyngeal-carriage survey and isolated the resistant strain from one asymptomatic carrier. Sequencing of the gene encoding subunit A of DNA gyrase (gyrA) revealed a mutation associated with fluoroquinolone resistance and suggests that the resistance was acquired by means of horizontal gene transfer with the commensal N. lactamica. In susceptibility testing of invasive N. meningitidis isolates from the Active Bacterial Core surveillance system between January 2007 and January 2008, an additional ciprofloxacin-resistant isolate was found, in this case from California. Ciprofloxacin-resistant N. meningitidis has emerged in North America.

Salmonella enterica pulsed-field gel electrophoresis clusters, Minnesota, USA, 2001-2007.

We determined characteristics of Salmonella enterica pulsed-field gel electrophoresis clusters that predict their being solved (i.e., that result in identification of a confirmed outbreak). Clusters were investigated by the Minnesota Department of Health by using a dynamic iterative model. During 2001-2007, a total of 43 (12.5%) of 344 clusters were solved. Clusters of ≥4 isolates were more likely to be solved than clusters of 2 isolates. Clusters in which the first 3 case isolates were received at the Minnesota Department of Health within 7 days were more likely to be solved than were clusters in which the first 3 case isolates were received over a period >14 days. If resources do not permit investigation of all S. enterica pulsed-field gel electrophoresis clusters, investigation of clusters of ≥4 cases and clusters in which the first 3 case isolates were received at a public health laboratory within 7 days may improve outbreak investigations.

Community-associated methicillin-resistant Staphylococcus aureus: trends in case and isolate characteristics from six years of prospective surveillance.

OBJECTIVES: In 2000, the Minnesota Department of Health (MDH) implemented active, sentinel site surveillance for community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA). Data from 2000-2005 were analyzed to determine trends in case characteristics, pulsed-field types (PFTs), and antimicrobial susceptibilities including inducible clindamycin resistance (ICR). METHODS: Active sentinel site surveillance was initiated in 2000 at 12 hospital laboratories that served inpatients and outpatients. Patient medical records were reviewed to determine if they met the epidemiologic case criteria for CA-MRSA; isolates were obtained from patients meeting these criteria. The MDH Public Health Laboratory performed pulsed-field gel electrophoresis subtyping and antimicrobial susceptibility testing, including ICR. RESULTS: The proportion of MRSA cases classified as CA increased from 11% to 33% (p<0.01). The proportion of cases with skin or soft tissue infections also increased compared with other infection types from 75% to 87% (p<0.01). During the surveillance period, USA300 replaced USA400 as the dominant PFT. With the change in dominant PFT, the proportion of isolates susceptible to erythromycin (45% to 13%, p<0.01) and ciprofloxacin (80% to 59%, p<0.01) decreased. The proportion of erythromycin-resistant/clindamycin-susceptible isolates with ICR (93% to 14%, p<0.01) decreased. The proportion of susceptible isolates also changed within the USA300 PFT; the proportion of isolates susceptible to erythromycin (33% vs. 3%) and the proportion susceptible to ciprofloxacin (67% to 62%) decreased significantly. CONCLUSION: CA-MRSA increased dramatically from 2000 to 2005. Changes in the predominant PFT have impacted susceptibility profiles of CA-MRSA, including ICR. Continued surveillance is needed to monitor the changing epidemiology of CA-MRSA and to inform clinical decisions.

Allele distribution and genetic diversity of VNTR loci in Salmonella enterica serotype Enteritidis isolates from different sources.

BACKGROUND: Salmonella enterica serotype Enteritidis (S. Enteritidis) is a zoonotic pathogen, which can be found in many sources including animals and the environment. However, little is known about the molecular relatedness among S. Enteritidis isolates from different sources. We have applied multiple-locus variable number tandem repeat analysis (MLVA) to study the genetic diversity of S. Enteritidis isolates from human and non-human sources. RESULTS: We identified 38 unique MLVA types using nine VNTR loci markers for discrimination between 145 S. Enteritidis isolates from different sources including humans (n = 41), chickens (n = 45), and eggs (n = 40). There were 20 distinct MLVA types identified from human isolates, 17 distinct MLVA types from chicken isolates, and 5 from egg isolates. We compared allele distribution and frequency for each VNTR marker and measured allelic polymorphism within each VNTR locus of S. Enteritidis isolates from the sources using Nei's diversity index (D). Differences in allele distribution and frequency were detected in most loci of study isolates. Different genetic diversity for certain loci was identified in isolates from different sources. The average of genetic diversity (D) was lower in egg isolates (0.16) compared to human (0.41) and chicken (0.30). However, for loci SE3, SE7, and SE9, human isolates showed significantly higher diversity than both chicken and egg isolates. Whereas for loci SE5 and SE10, chicken isolates had significantly higher diversity than both human and egg isolates. Minimum-spanning tree (MST) comprised one major cluster, a minor cluster, and four clonal expansions. MLVA application enabled a cluster analysis by the MST of the S. Enteritidis isolates by sources, which allows a great insight into the genetic relatedness and the possible flow of these organisms between different reservoirs and humans. CONCLUSION: Differences in allele distribution and genetic diversity of VNTR loci in S. Enteritidis isolates from different sources were found. Polymorphism in most of the VNTR loci was more frequent among human S. Enteritidis isolates than isolates from chickens or eggs. Therefore, VNTR profiles of S. Enteritidis isolates from a specific source should be further evaluated as potential markers in epidemiologic investigations to trace S. Enteritidis to their probable source.

Outbreaks of salmonellosis in Minnesota (1998 through 2006) associated with frozen, microwaveable, breaded, stuffed chicken products.

From 1998 through 2006, four outbreaks of salmonellosis associated with raw, frozen, microwaveable, breaded, prebrowned, stuffed chicken products were identified in Minnesota. In 1998, 33 Salmonella Typhimurium cases were associated with a single brand of Chicken Kiev. In 2005, four Salmonella Heidelberg cases were associated with a different brand and variety (Chicken Broccoli and Cheese). From 2005 to 2006, 27 Salmonella Enteritidis cases were associated with multiple varieties of product, predominately of the same brand involved in the 1998 outbreak. In 2006, three Salmonella Typhimurium cases were associated with the same brand of product involved in the 2005 Salmonella Heidelberg outbreak. The outbreak serotype and pulsed-field gel electrophoresis subtype of Salmonella were isolated from product in each outbreak. In these outbreaks, most individuals affected thought that the product was precooked due to its breaded and prebrowned nature, most used a microwave oven, most did not follow package cooking instructions, and none took the internal temperature of the cooked product. Similar to previous salmonellosis outbreaks associated with raw, breaded chicken nuggets or strips in Canada and Australia, inadequate labeling, consumer responses to labeling, and microwave cooking were the key factors in the occurrence of these outbreaks. Modification of labels, verification of cooking instructions by the manufacturer, and notifications to alert the public that these products contain raw poultry, implemented because of the first two outbreaks, did not prevent the other outbreaks. Microwave cooking is not recommended as a preparation method for these types of products, unless they are precooked or irradiated prior to sale.

Multiple-locus variable-number tandem repeat analysis of Salmonella Enteritidis isolates from human and non-human sources using a single multiplex PCR.

Simplified multiple-locus variable-number tandem repeat analysis (MLVA) was developed using one-shot multiplex PCR for seven variable-number tandem repeats (VNTR) markers with high diversity capacity. MLVA, phage typing, and PFGE methods were applied on 34 diverse Salmonella Enteritidis isolates from human and non-human sources. MLVA detected allelic variations that helped to classify the S. Enteritidis isolates into more evenly distributed subtypes than other methods. MLVA-based S. Enteritidis clonal groups were largely associated with sources of the isolates. Nei's diversity indices for polymorphism ranged from 0.25 to 0.70 for seven VNTR loci markers. Based on Simpson's and Shannon's diversity indices, MLVA had a higher discriminatory power than pulsed field gel electrophoresis (PFGE), phage typing, or multilocus enzyme electrophoresis. Therefore, MLVA may be used along with PFGE to enhance the effectiveness of the molecular epidemiologic investigation of S. Enteritidis infections.

Emergence of multidrug-resistant Salmonella enterica serotype Newport in Minnesota.

We report a concurrent increase in the number of isolates of Salmonella enterica serotype Newport and the rate of multidrug resistance in S. Newport isolates from animal and human populations in Minnesota. Antimicrobial susceptibility and pulsed-field gel electrophoresis analysis demonstrated heterogeneity of isolates and showed that 1 pulsed-field gel electrophoresis cluster contained most of the multidrug-resistant isolates with a resistance pattern and most class 1 integron isolates, implying the clonal origin of the isolates.

A community outbreak of conjunctivitis caused by nontypeable Streptococcus pneumoniae in Minnesota.

BACKGROUND: The Minnesota Department of Health (MDH) was notified of an outbreak of conjunctivitis in city A with cultures positive for Streptococcus pneumoniae. METHODS: MDH staff contacted clinics and schools in city A and city B regarding conjunctivitis cases, reviewed clinical findings of conjunctivitis cases in city A and collected isolates for subtyping. RESULTS: Between September 1 and December 12, 2003, cities A and B reported 735 conjunctivitis cases. Fifty-one percent of the cases were reported from schools, childcare centers and colleges. Adults were more likely to report itching, burning or swelling of the eye(s); children were more likely to report crusty eyes (P < 0.05). Forty-nine percent of conjunctival cultures (71 of 144) were positive for S. pneumoniae. All isolates were nontypeable by serotyping. Pulsed field gel electrophoresis identified 3 clonal groups with 84% of isolates belonging to one clonal group. Multilocus sequence typing revealed that isolates had the same multilocus sequence type as isolates from a 2002 outbreak at a New England college. CONCLUSIONS: This outbreak was widespread in the community and conjunctivitis clinical presentation varied by age. The predominant strains in this outbreak were related to a pneumococcal strain implicated in prior conjunctivitis outbreaks, suggesting these strains have a predilection for causing conjunctivitis.

Outbreak of Escherichia coli O157:H7 infections associated with nonintact blade-tenderized frozen steaks sold by door-to-door vendors.

Steaks have not been recognized as an important vehicle of Escherichia coli O157:H7 infection. During 11 to 27 June 2003, the Minnesota Department of Health (MDH) identified four O157 infection cases with the same pulsed-field gel electrophoresis (PFGE) subtype. All four case patients consumed brand A vacuum packed frozen steaks sold by door-to-door vendors. The steaks were blade tenderized and injected with marinade (i.e., nonintact). Information from single case patients in Michigan and Kansas identified through PulseNet confirmed the outbreak. The MDH issued a press release on 27 June to warn consumers, prompting a nationwide recall of 739,000 lb (335,506 kg) of frozen beef products. The outbreak resulted in six culture-confirmed cases (including one with hemolytic uremic syndrome) and two probable cases in Minnesota and single confirmed cases in four other states. The outbreak PFGE subtype of O157 was isolated from unopened brand A bacon-wrapped fillets from five affected Minnesota households. A fillet from one affected household was partially cooked in the laboratory, and the same O157 subtype was isolated from the uncooked interior. The tenderizing and injection processes likely transferred O157 from the surface to the interior of the steaks. These processing methods create new challenges for prevention of O157 infection. Food regulatory officials should reevaluate safety issues presented by nonintact steak products, such as microbiologic hazards of processing methods, possible labeling to distinguish intact from nonintact steaks, and education of the public and commercial food establishments on the increased risk associated with undercooked nonintact steaks. Information on single cases of O157 infection in individual states identified through PulseNet can be critical in solving multistate outbreaks in a timely manner.

USA300 and USA500 clonal lineages of Staphylococcus aureus do not produce a capsular polysaccharide due to conserved mutations in the cap5 locus.

UNLABELLED: The surface capsular polysaccharide (CP) is a virulence factor that has been used as an antigen in several successful vaccines against bacterial pathogens. A vaccine has not yet been licensed against Staphylococcus aureus, although two multicomponent vaccines that contain CP antigens are in clinical trials. In this study, we evaluated CP production in USA300 methicillin-resistant S. aureus (MRSA) isolates that have become the predominant community-associated MRSA clones in the United States. We found that all 167 USA300 MRSA and 50 USA300 methicillin-susceptible S. aureus (MSSA) isolates were CP negative (CP(-)). Moreover, all 16 USA500 isolates, which have been postulated to be the progenitor lineage of USA300, were also CP(-). Whole-genome sequence analysis of 146 CP(-) USA300 MRSA isolates revealed they all carry a cap5 locus with 4 conserved mutations compared with strain Newman. Genetic complementation experiments revealed that three of these mutations (in the cap5 promoter, cap5D nucleotide 994, and cap5E nucleotide 223) ablated CP production in USA300 and that Cap5E75 Asp, located in the coenzyme-binding domain, is essential for capsule production. All but three USA300 MSSA isolates had the same four cap5 mutations found in USA300 MRSA isolates. Most isolates with a USA500 pulsotype carried three of these four USA300-specific mutations, suggesting the fourth mutation occurred in the USA300 lineage. Phylogenetic analysis of the cap loci of our USA300 isolates as well as publicly available genomes from 41 other sequence types revealed that the USA300-specific cap5 mutations arose sequentially in S. aureus in a common ancestor of USA300 and USA500 isolates. IMPORTANCE: The USA300 MRSA clone emerged as a community-associated pathogen in the United States nearly 20 years ago. Since then, it has rapidly disseminated and now causes health care-associated infections. This study shows that the CP-negative (CP(-)) phenotype has persisted among USA300 isolates and is a universal and characteristic trait of this highly successful MRSA lineage. It is important to note that a vaccine consisting solely of CP antigens would not likely demonstrate high efficacy in the U.S. population, where about half of MRSA isolates comprise USA300. Moreover, conversion of a USA300 strain to a CP-positive (CP(+)) phenotype is unlikely in vivo or in vitro since it would require the reversion of 3 mutations. We have also established that USA300 MSSA isolates and USA500 isolates are CP(-) and provide new insight into the evolution of the USA300 and USA500 lineages.

Vancomycin-intermediate Staphylococcus aureus with phenotypic susceptibility to methicillin in a patient with recurrent bacteremia.

Vancomycin-intermediate Staphylococcus aureus (VISA) are an emerging problem. We observed a statistically significant inverse relationship in the MICs of vancomycin and oxacillin in S. aureus isolates from a patient undergoing hemodialysis who received 26 weeks of treatment with vancomycin during November 1999 through April 2000. All isolates were mecA positive and were indistinguishable by pulsed-field gel electrophoresis. The evolving susceptibility patterns of this strain highlight the challenges of detecting and treating VISA infections.

Concurrent outbreaks of Shigella sonnei and enterotoxigenic Escherichia coli infections associated with parsley: implications for surveillance and control of foodborne illness.

In recent years, the globalization of the food supply and the development of extensive food distribution networks have increased the risk of foodborne disease outbreaks involving multiple states or countries. In particular, outbreaks associated with fresh produce have emerged as an important public health concern. During July and August 1998, eight restaurant-associated outbreaks of shigellosis caused by a common strain of Shigella sonnei occurred in the United States and Canada. The outbreak strain was characterized by unique pulsed-field gel electrophoresis patterns. Epidemiologic investigation determined that the illness was associated with the ingestion of parsley at four restaurants; at the other four restaurants, the majority of the people who contracted the illness ate parsley. Isolates from patrons in two unrelated restaurant-associated enterotoxigenic Escherichia coli (ETEC) outbreaks in Minnesota shared a common serotype and pulsed-field gel electrophoresis (PFGE) pattern. Parsley was the implicated or suspected source of both ETEC outbreaks. In each of the outbreak-associated restaurants, parsley was chopped, held at room temperature, and used as an ingredient or garnish for multiple dishes. Infected food workers at several restaurants may also have contributed to the propagation of the outbreak. The sources of parsley served in outbreak-associated restaurants were traced, and a 1,600-acre farm in Baja California, Mexico, was identified as a likely source of the parsley implicated in six of the seven Shigella outbreaks and as a possible source of the parsley implicated in the two ETEC outbreaks. Global food supplies and large distribution networks demand strengthened laboratory and epidemiologic capacity to enable state and local public health agencies to conduct foodborne disease surveillance and to promote effective responses to multistate outbreaks.

Comparison of community- and health care-associated methicillin-resistant Staphylococcus aureus infection.

CONTEXT: Methicillin-resistant Staphylococcus aureus (MRSA) has traditionally been considered a health care-associated pathogen in patients with established risk factors. However, MRSA has emerged in patients without established risk factors (community-associated MRSA). OBJECTIVE: To characterize epidemiological and microbiological characteristics of community-associated MRSA cases compared with health care-associated MRSA cases. DESIGN, SETTING, AND PATIENTS: Prospective cohort study of patients with MRSA infection identified at 12 Minnesota laboratory facilities from January 1 through December 31, 2000, comparing community-associated (median age, 23 years) with health care-associated (median age, 68 years) MRSA cases. MAIN OUTCOME MEASURES: Clinical infections associated with either community-associated or health care-associated MRSA, microbiological characteristics of the MRSA isolates including susceptibility testing, pulsed-field gel electrophoresis, and staphylococcal exotoxin gene testing. RESULTS: Of 1100 MRSA infections, 131 (12%) were community-associated and 937 (85%) were health care-associated; 32 (3%) could not be classified due to lack of information. Skin and soft tissue infections were more common among community-associated cases (75%) than among health care-associated cases (37%) (odds ratio [OR], 4.25; 95% confidence interval [CI], 2.97-5.90). Although community-associated MRSA isolates were more likely to be susceptible to 4 antimicrobial classes (adjusted OR, 2.44; 95% CI, 1.35-3.86), most community-associated infections were initially treated with antimicrobials to which the isolate was nonsusceptible. Community-associated isolates were also more likely to belong to 1 of 2 pulsed-field gel electrophoresis clonal groups in both univariate and multivariate analysis. Community-associated isolates typically possessed different exotoxin gene profiles (eg, Panton Valentine leukocidin genes) compared with health care-associated isolates. CONCLUSIONS: Community-associated and health care-associated MRSA cases differ demographically and clinically, and their respective isolates are microbiologically distinct. This suggests that most community-associated MRSA strains did not originate in health care settings, and that their microbiological features may have contributed to their emergence in the community. Clinicians should be aware that therapy with beta-lactam antimicrobials can no longer be relied on as the sole empiric therapy for severely ill outpatients whose infections may be staphylococcal in origin.

Pre- and postvaccination clonal compositions of invasive pneumococcal serotypes for isolates collected in the United States in 1999, 2001, and 2002.

Monitoring of serotypes and their clonal associations is critical as pneumococci adapt to the selective pressures exerted by the pneumococcal seven-valent conjugate vaccine (PCV7). We genotyped 1,476 invasive isolates from the Active Bacterial Core surveillance (705 [89.8%] of the isolates were obtained from children <5 years of age, and 771 [18.4%] of the isolates were obtained from individuals >5 years of age) in 2001 and 2002 (after the introduction of PCV7). The data were compared to the results for 1,168 invasive isolates (855 [83.9%] of the isolates were from children <5 years of age) collected in 1999. Among children <5 years of age, the incidence of invasive disease due to non-PCV7 serogroups together with serogroup 19A increased (P < 0.001). Eighty-three clonal sets, representing 177 multilocus sequence types (STs), were compiled from the 3-year isolate set. Among the non-PCV7 serogroups, newly emerging clones were uncommon; and a significant expansion of already established clones occurred for serotypes 3 (ST180), 7F (ST191), 15BCF (ST199), 19A (ST199), 22F (ST433), 33F (ST662), and 38 (ST393). However, additional minor clonal types within serotypes 1, 6A, 6B, 7C, 9N, 10A, 12F, 14, 15B/C, 17F, 19A, 19F, 20, 22F, and 33F that were absent in 1999 were found during 2001 and 2002. Although 23 clonal sets exhibited multiple serotypes, for most serotypes there were either no changes or modest changes in clonal compositions since the introduction of PCV7. The only example of an identical ST shared between non-PCV7 and PCV7 or PCV7-related serotypes was ST199; however, ST199 was prevalent within serotypes 15B/C and 19A before and after PCV7 introduction. Continued genotypic surveillance is warranted, since certain clones not targeted by PCV7 are expanding, and their emergence as significant pathogens could occur with maintained vaccine pressure.

Antimicrobial-drug susceptibility of human and animal Salmonella typhimurium, Minnesota, 1997-2003.

We compared antimicrobial resistance phenotypes and pulsed-field gel electrophoresis (PFGE) subtypes of 1,028 human and 716 animal Salmonella enterica serotype Typhimurium isolates from Minnesota from 1997 to 2003. Overall, 29% of human isolates were multidrug resistant. Predominant phenotypes included resistance to ampicillin, chloramphenicol or kanamycin, streptomycin, sulfisoxazole, and tetracycline (ACSSuT or AKSSuT). Most human multidrug-resistant isolates belonged to PFGE clonal group A, characterized by ACSSuT resistance (64%), or clonal group B, characterized by AKSSuT resistance (19%). Most animal isolates were from cattle (n = 358) or swine (n = 251). Eighty-one percent were multidrug resistant; of these, 54% were at least resistance phenotype ACSSuT, and 43% were at least AKSSuT. More than 80% of multidrug-resistant isolates had a clonal group A or B subtype. Resistance to ceftriaxone and nalidixic acid increased, primarily among clonal group A/ACSSuT isolates. Clonal group B/AKSSuT isolates decreased over time. These data support the hypothesis that food animals are the primary reservoir of multidrug-resistant S. Typhimurium.

Community-associated methicillin-resistant Staphylococcus aureus, Minnesota, 2000-2003.

We compared characteristics of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) skin and soft tissue infections (SSTIs) and CA-MRSA invasive disease identified in Minnesota from 2000 through 2003. A total of 586 patients with SSTIs and 65 patients with invasive disease were identified. Patients with invasive disease were more likely to be smokers (p = 0.03), and report a history of immunosuppressive therapy (p = 0.03), emphysema (p = 0.011), or injection drug use (p = 0.020) than were SSTI patients. Invasive disease isolates were less likely to be susceptible to ciprofloxacin (p = 0.002) and clindamycin (p = 0.001) and more likely to have healthcare-associated pulsed-field gel electrophoresis subtypes than SSTI isolates (p<0.001). Patients with invasive disease may have had healthcare exposures that put them at risk of acquiring healthcare-associated MRSA and which were not exclusion criteria in the CA-MRSA case definition. Continued surveillance of MRSA is needed to better characterize CA-MRSA infections.

Outbreak of osteomyelitis/septic arthritis caused by Kingella kingae among child care center attendees.

OBJECTIVE: Kingella kingae often colonizes the oropharyngeal and respiratory tracts of children but infrequently causes invasive disease. In mid-October 2003, 2 confirmed and 1 probable case of K kingae osteomyelitis/septic arthritis occurred among children in the same 16- to 24-month-old toddler classroom of a child care center. The objective of this study was to investigate the epidemiology of K kingae colonization and invasive disease among child care attendees. METHODS: Staff at the center were interviewed, and a site visit was performed. Oropharyngeal cultures were obtained from the staff and children aged 0 to 5 years to assess the prevalence of Kingella colonization. Bacterial isolates were subtyped by pulsed-field gel electrophoresis (PFGE), and DNA sequencing of the 16S rRNA gene was performed. A telephone survey inquiring about potential risk factors and the general health of each child was also conducted. All children and staff in the affected toddler classroom were given rifampin prophylaxis and recultured 10 to 14 days later. For epidemiologic and microbiologic comparison, oropharyngeal cultures were obtained from a cohort of children at a control child care center with similar demographics and were analyzed using the same laboratory methods. The main outcome measures were prevalence and risk factors for colonization and invasive disease and comparison of bacterial isolates by molecular subtyping and DNA sequencing. RESULTS: The 2 confirmed case patients required hospitalization, surgical debridement, and intravenous antibiotic therapy. The probable case patient was initially misdiagnosed; MRI 16 days later revealed evidence of ankle osteomyelitis. The site visit revealed no obvious outbreak source. Of 122 children in the center, 115 (94%) were cultured. Fifteen (13%) were colonized with K kingae, with the highest prevalence in the affected toddler classroom (9 [45%] of 20 children; all case patients tested negative but had received antibiotics). Six colonized children were distributed among the older classrooms; 2 were siblings of colonized toddlers. No staff (n = 28) or children aged <16 months were colonized. Isolates from the 2 confirmed case patients and from the colonized children had an indistinguishable PFGE pattern. No risk factors for invasive disease or colonization were identified from the telephone survey. Of the 9 colonized toddlers who took rifampin, 3 (33%) remained positive on reculture; an additional toddler, initially negative, was positive on reculture. The children of the control child care center demonstrated a similar degree and distribution of K kingae colonization; of 118 potential subjects, 45 (38%) underwent oropharyngeal culture, and 7 (16%) were colonized with K kingae. The highest prevalence again occurred in the toddler classrooms. All 7 isolates from the control facility had an indistinguishable PFGE pattern; this pattern differed from the PFGE pattern observed from the outbreak center isolates. 16S rRNA gene sequencing demonstrated that the outbreak K kingae strain exhibited >98% homology to the ATCC-type strain, although several sequence deviations were present. Sequencing of the control center strain demonstrated more homology to the outbreak center strain than to the ATCC-type strain. CONCLUSIONS: This is the first reported outbreak of invasive K kingae disease. The high prevalence in the affected toddler class and the matching PFGE pattern are consistent with child-to-child transmission within the child care center. Rifampin was modestly effective in eliminating carriage. DNA sequence analysis suggests that there may be considerable variability within the species K kingae and that different K kingae strains may demonstrate varying degrees of pathogenicity.

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.

Emergence of a novel penicillin-nonsusceptible, invasive serotype 35B clone of Streptococcus pneumoniae within the United States.

Monitoring antibiotic-resistant pneumococcal strains not covered by the 7-valent conjugate vaccine is an important priority. The Centers for Disease Control and Prevention's Active Bacterial Core Surveillance identified 68 invasive penicillin-nonsusceptible serotype 35B (PN35B) isolates recovered from 1995 to 2001 from patients residing in the states of California, Colorado, Connecticut, Georgia, Maryland, Minnesota, New York, Oregon, Tennessee, and Texas. Nonsusceptible isolates accounted for 69% of all serotype 35B isolates recovered during this time. Twelve (18%) of the 68 PN35B isolates recovered since 1995 were obtained from pediatric patients. These 68 isolates exhibited penicillin MICs of 0.25-2 microg/mL and reduced susceptibility to cefotaxime. Representative PN35B isolates exhibited a common chromosomal macrorestriction profile and identical penicillin-binding-protein gene restriction profiles characteristic of penicillin-resistant strains, and they shared a unique 7-locus sequence type that included 3 new alleles. The mosaic pbp2b and divergent ddl sequences were suggestive of interspecies recombination at the ddl-pbp2b chromosomal region.

Multilocus sequence typing reveals a lack of diversity among Escherichia coli O157:H7 isolates that are distinct by pulsed-field gel electrophoresis.

Escherichia coli O157:H7 is a major cause of foodborne illness in the United States. Pulsed-field gel electrophoresis (PFGE) is the molecular epidemiologic method mostly commonly used to identify food-borne outbreaks. Although PFGE is a powerful epidemiologic tool, it has disadvantages that make a DNA sequence-based approach potentially attractive. Multilocus sequence typing (MLST) analyzes the internal fragments of housekeeping genes to establish genetic relatedness between isolates. We sequenced selected portions of seven housekeeping genes and two membrane protein genes (ompA and espA) of 77 isolates that were diverse by PFGE to determine whether there was sufficient sequence variation to be useful as an epidemiologic tool. There was no DNA sequence diversity in the sequenced portions of the seven housekeeping genes and espA. For ompA, all but five isolates had sequence identical to that of the reference strains. E. coli O157:H7 has a striking lack of genetic diversity in the genes we explored, even among isolates that are clearly distinct by PFGE. Other approaches to identify improved molecular subtyping methods for E. coli 0157:H7 are needed.