Clonal diversity and turnover of Streptococcus mitis bv. 1 on shedding and nonshedding oral surfaces of human infants during the first year of life

Streptococcus mitis bv. 1 is a pioneer colonizer of the human oral cavity. Studies of its population dynamics within parents and their infants and within neonates have shown extensive diversity within and between subjects. We examined the genetic diversity and clonal turnover of S. mitis bv. 1 isolated from the cheeks, tongue, and primary incisors of four infants from birth to 1 year of age. In addition, we compared the clonotypes of S. mitis bv. 1 isolated from their mothers' saliva collected in parallel to determine whether the mother was the origin of the clones colonizing her infant. Of 859 isolates obtained from the infants, 568 were unique clones. Each of the surfaces examined, whether shedding or nonshedding, displayed the same degree of diversity. Among the four infants it was rare to detect the same clone colonizing more than one surface at a given visit. There was little evidence for persistence of clones, but when clones were isolated on multiple visits they were not always found on the same surface. A similar degree of clonal diversity of S. mitis bv. 1 was observed in the mothers' saliva as in their infants' mouths. Clones common to both infant and mothers' saliva were found infrequently suggesting that this is not the origin of the infants' clones. It is unclear whether mucosal immunity exerts the environmental pressure driving the genetic diversity and clonal turnover of S. mitis bv. 1, which may be mechanisms employed by this bacterium to evade immune elimination.     

Glucose Uptake by Streptococcus mutans, Streptococcus mitis, and Actinomyces viscosus in the Presence of Human Saliva

Glucose uptake was examined by using whole-cell suspensions of Streptococcus mutans (strains BHT, Ingbritt, and GS-5), Streptococcus mitis (strains 9811 and 72×41), and Actinomyces viscosus (strains T6 and WVU626) incubated for up to 90 min in 0 to 82% (vol/vol) human whole salivary supernatant. Glucose uptake by the S. mutans strains was completely inhibited at all saliva concentrations. Dithiothreitol (DTT), present during saliva incubation, prevented saliva inhibition. Glucose uptake was also restored when saliva-inhibited cells were subsequently exposed to DTT. The inclusion of catalase in the saliva incubation mixtures resulted in protection equal to that obtained with DTT. The S. mitis strains were also inhibited by saliva but to a far lesser extent that S. mutans. DTT and catalase also protected S. mitis from saliva inhibition. Both A. viscosus strains were completely refractory to saliva inhibition of glucose uptake. Based on (i) the sensitivity of the catalase-negative streptococci and the resistance of catalase-positive actinomyces to saliva inhibition and (ii) the equal and complete protection to saliva inhibition afforded by DTT and catalase, we conclude that the lactoperoxidase-SCN⁻-H₂O₂ system in saliva was the only antibacterial system expressed under our experimental conditions. The relative resistance of S. mitis 9811 (compared with S. mutans BHT) to saliva inhibition was shown not to result from poor H₂O₂ production in either glucose-supplemented buffer or saliva solutions. S. mitis produced inhibitory quantities of H₂O₂ that equaled or exceeded S. mutans H₂O₂ accumulation. It is suggested that S. mitis might possess a greater ability to repair lactoperoxidase-mediated damage than does S. mutans. Every organism studied exhibited a saliva concentration-dependent, cell growth-independent stimulation of glucose uptake after 60 to 90 min of incubation. The A. viscosus and S. mitis strains showed saliva stimulation (or stabilization) of glucose uptake with unsupplemented saliva. In the case of S. mutans, saliva stimulation was only observed when DTT was present. The possible role of salivary lactoperoxidase as a modulator of the intraoral site specificities exhibited by S. mutans is discussed.     

Effect of Human Saliva on Glucose Uptake by Streptococcus mutans and Other Oral Microorganisms

We examined the effects of human whole salivary supernatant and parotid fluid on glucose uptake by Streptococcus mutans, Streptococcus sanguis, Streptococcus mitis, Actinomyces viscosus, Staphylococcus aureus, and Escherichia coli. The following three effects of saliva were observed: (i) inhibition of glucose uptake (S. mutans, S. sanguis), (ii) promotion of a transient, rapid (0 to 30 s) burst of glucose uptake (S. mutans, S. sanguis), and (iii) enhancement of glucose uptake (S. mitis, A. viscosus, S. aureus, E. coli). We observed no differences between the effects of whole salivary supernatant and the effects of parotid fluid. Heat treatment (80°C, 10 min) of saliva or the addition of dithiothreitol abolished inhibition of glucose uptake. Supplementation of saliva with H₂O₂ potentiated inhibition of glucose uptake. S. mitis and A. viscosus, which were stimulated by saliva alone, were inhibited by H₂O₂-supplemented saliva; 50% inhibition of glucose uptake by S. mutans and S. mitis required ca. 10 μM H₂O₂ in 50% (vol/vol) saliva. Loss of the inhibitory action of saliva occurred at about 5% (vol/vol) saliva. Supplementation of saliva dilutions with SCN⁻ and H₂O₂ extended the inhibitory activity to solutions containing ca. 0.2% (vol/vol) saliva. We suggest that the salivary lactoperoxidase-SCN⁻-H₂O₂ system is responsible for the inhibitory activity of saliva reported here. Furthermore, we concluded that lactoperoxidase and SCN⁻ are present in saliva specimens in concentrations that exceed minimal inhibitory levels by factors of ca. 500 and 10 to 20, respectively. The resistance of A. viscosus, S. aureus, and E. coli to the inhibitory potential of saliva alone was probably due to the production of catalase by these organisms. The resistance of S. mitis may have been due to special effects of saliva on H₂O₂ accumulation by this organism compared with S. mutans and S. sanguis. The basis of saliva-dependent enhancement of glucose uptake and the basis of promotion of a transient, rapid burst of glucose uptake are unknown. The role of the salivary lactoperoxidase-SCN⁻-H₂O₂ system in the oral microbial ecosystem is discussed.     

Relationship between oral anaerobic bacteria and otitis media with effusion

Objective: In this study hypothesing the translocation of oral bacteria from oropharynx into the middle ear cavity may be involved in the pathogenesis of otitis media with effusion (OME), we aimed to investigate the presence and similarity of Fusobacterium nucleatum and Treponema denticola in saliva, nasopharyngeal secretion and the middle ear effusion samples from the children with OME.Methods: Totally 20 children with OME undergoing myringotomy and ventilation tube placement were attended. Stimulated saliva samples were collected after otorhinolaryngological and oral examinations were done. The middle ear effusion and nasopharyngeal secretions were collected during the operations. The presence of F. nucleatum and T. denticola were detected using 16SrRNA-based PCR. The clonal similarities of the bacteria were detected in the samples which the same bacteria had been detected in each samples of the same child. After DNA sequencing, clonal similarity was determined by 16SrRNA gene clone library analysis. The sequences from each clone were compared with similar sequences of reference organisms by FASTA search.Results: T. denticola was detected only in four (20%) saliva and in one (5%) nasopharyngeal sample. F. nucleatum was detected in 11 (55%) saliva, eight (40%) nasopharyngeal and six (30%) middle ear effusion samples. Sequences from F.nucleatum clones derived from three different anatomic sites within patients were similar in 33% of OME patients, indicating their genetic relatedness.Conclusions: Bacteria involved in this process most likely originate from the oropharynx since they show a close genetic relatedness with their oropharyngeal counterparts.     

Study of Humoral Immunity to Commensal Oral Bacteria in Human Infants Demonstrates the Presence of Secretory Immunoglobulin A Antibodies Reactive with Actinomyces naeslundii Genospecies 1 and 2 Ribotypes

The mouths of three human infants were examined from birth to age 2 years to detect colonization of Actinomyces naeslundii genospecies 1 and 2. These bacteria did not colonize until after tooth eruption. The diversity of posteruption isolates was determined by ribotyping. Using immunoblotting and enzyme-linked immunosorbent assay, we determined the reactivity of secretory immunoglobulin A (SIgA) antibodies in saliva samples collected from each infant before and after colonization against cell wall proteins from their own A. naeslundii strains and carbohydrates from standard A. naeslundii genospecies 1 and 2 strains. A. naeslundii genospecies 1 and 2 carbohydrate-reactive SIgA antibodies were not detected in any saliva sample. However, SIgA antibodies reactive with cell wall proteins were present in saliva before these bacteria colonized the mouth. These antibodies could be almost completely removed by absorption with A. odontolyticus, a species known to colonize the human mouth shortly after birth. However, after colonization by A. naeslundii genospecies 1 and 2, specific antibodies were induced that could not be removed by absorption with A. odontolyticus. Cluster analysis of the patterns of reactivity of postcolonization salivary antibodies from each infant with antigens from their own strains showed that not only could these antibodies discriminate among strains but antibodies in saliva samples collected at different times showed different reactivity patterns. Overall, these data suggest that, although much of the salivary SIgA antibodies reactive with A. naeslundii genospecies 1 and 2 are directed against genus-specific or more broadly cross-reactive antigens, species, genospecies, and possibly strain-specific antibodies are induced in response to colonization.     

Molecular Types of Methicillin-Resistant Staphylococcus aureus and Methicillin-Sensitive S. aureus Strains Causing Skin and Soft Tissue Infections and Nasal Colonization,Identified in Community Health Centers in New York City

In November 2011, The Rockefeller University Center for Clinical and Translational Science (CCTS), the Laboratory of Microbiology and Infectious Diseases, and Clinical Directors Network (CDN) launched a research and learning collaborative project with six community health centers in the New York City metropolitan area to determine the nature (clonal type) of community-acquired Staphylococcus aureus strains causing skin and soft tissue infections (SSTIs). Between November 2011 and March 2013, wound and nasal samples from 129 patients with active SSTIs suspicious for S. aureus were collected and characterized by molecular typing techniques. In 63 of 129 patients, the skin wounds were infected by S. aureus: methicillin-resistant S. aureus (MRSA) was recovered from 39 wounds and methicillin-sensitive S. aureus (MSSA) was recovered from 24. Most—46 of the 63–wound isolates belonged to the CC8/Panton-Valentine leukocidin-positive (PVL⁺) group of S. aureus clone USA300: 34 of these strains were MRSA and 12 were MSSA. Of the 63 patients with S. aureus infections, 30 were also colonized by S. aureus in the nares: 16 of the colonizing isolates were MRSA, and 14 were MSSA, and the majority of the colonizing isolates belonged to the USA300 clonal group. In most cases (70%), the colonizing isolate belonged to the same clonal type as the strain involved with the infection. In three of the patients, the identity of invasive and colonizing MRSA isolates was further documented by whole-genome sequencing.     

Denaturing Gradient Gel Electrophoresis of PCR-Amplified gki Genes: a New Technique for Tracking Streptococci

Viridans group streptococci (VGS) are a well-known cause of infections in immunocompromised patients, accounting for severe morbidity and mortality. Streptococcus mitis group species (Streptococcus mitis, Streptococcus pneumoniae, Streptococcus oralis) are among the VGS most often encountered in clinical practice. Identifying the portal of entry for S. mitis group strains is crucial for interventions preventing bacterial translocation. Unfortunately, tracking the source of S. mitis group strains is dependent on a combination of extremely laborious and time-consuming cultivation and molecular techniques (enterobacterial repetitive intergenic consensus-PCR [ERIC-PCR]). To simplify this procedure, a PCR analysis with newly designed primers targeting the household gene glucose kinase (gki) was used in combination with denaturing gradient gel electrophoresis (DGGE). This gki-PCR-DGGE technique proved to be specific for S. mitis group strains. Moreover, these strains could be detected in samples comprised of highly diverse microbiota, without prior cultivation. To study the feasibility of this new approach, a pilot study was performed. This confirmed that the source of S. mitis group bacteremia in pediatric patients with acute myeloid leukemia could be tracked back to the throat in five out of six episodes of bacteremia, despite the fact that throat samples are polymicrobial samples containing multiple S. mitis group strains. In contrast, using the classical combination of cultivation techniques and ERIC-PCR, we could detect these strains in only two out of six cases, showing the superiority of the newly developed technique. The new gki-PCR-DGGE technique can track the source of S. mitis group strains in polymicrobial samples without prior cultivation. Therefore, it is a valuable tool in future epidemiological studies.Viridans group streptococci (VGS) are a notorious cause of bacteremia in immunocompromised cancer patients (1, 3, 5). In pediatric patients with acute myeloid leukemia (AML), up to 62.5% of all infections are caused by VGS, accounting for serious morbidity and mortality (8, 18).The group of VGS is divided into 24 different species, of which Streptococcus mitis is one of the most encountered in clinical practice. Compared to the other 23 VGS species, S. mitis is associated with higher levels of bacteremia, a higher risk of infectious complications, and more resistance to antibiotics in cancer patients (5, 9).Despite the clinical relevance of S. mitis infections, the portal of entry for these bacteria remains questionable. However, to study the effect of interventions to prevent S. mitis translocation, it is essential to know the source of these disease-causing streptococci. Comparison of streptococcal strains from different sampling sites of the human body is crucial to detect the portal of entry for these VGS. This comparison is hindered by the fact that the throat, and to a lesser extent the large intestine, is inhabited by many different VGS strains, between which it is hard to discriminate. Moreover, strain detection is dependent on a combination of laborious culturing techniques and molecular typing. Furthermore, due to variable and inconclusive biochemical reactions, it is often difficult to name VGS to the species level, let alone to the strain level. Especially the differentiation between S. mitis, Streptococcus oralis, and Streptococcus pneumoniae, further referred to as the S. mitis group, has been proven to be very hard and has led to the introduction of DNA-based approaches such as multilocus sequence typing (7, 14). However, to investigate the source of S. mitis group strains causing infections, a method of discriminating S. mitis group members at the strain level is needed. Using conventional culturing techniques combined with molecular typing, it is nearly impossible to compare S. mitis group strains isolated from surveillance samples of various locations, as it is very laborious. Until now, no culture-independent molecular detection technique exists that directly discriminates between S. mitis group strains.In this paper, we describe a new gki-PCR-denaturing gradient gel electrophoresis (DGGE) technique consisting of specific PCR primers, targeting the household gene glucose kinase (gki), used in combination with DGGE to discriminate between different S. mitis group strains. To test the feasibility of this new approach, we compared S. mitis group strains isolated from blood cultures with strains in throat swabs, gastric fluids, and fecal samples in immunocompromised AML patients, independent of culturing techniques and biochemical analyses. We demonstrate that this new gki-PCR-DGGE technique can be used to detect S. mitis group strains in surveillance samples, which may contain a high level of bacterial diversity. Moreover, this new technique can be used to track the source of invasive S. mitis group bacteria.     

Simultaneous Detection of Streptococcus pneumoniae,S. mitis,and S. oralis by a Novel Multiplex PCR Assay Targeting the gyrB Gene

A multiplex PCR (mPCR) protocol was developed for simultaneous detection of the gyrB gene in Streptococcus pneumoniae, Streptococcus mitis, and Streptococcus oralis, and the specificity was evaluated using 141 coccus strains. Genomic DNAs purified from S. pneumoniae, S. mitis, and S. oralis strains were efficiently detected with size differences, whereas no PCR products were amplified from any of the reference strains tested. A pilot study of 47 human oral swab specimens was conducted in parallel, and the mPCR assay identified S. pneumoniae in 1 sample, S. mitis in 8 samples, and S. oralis in 2 samples, providing a powerful means for characterization at the level of species compared with traditional culture analysis. Our results suggest that the mPCR protocol presented here is a sensitive and promising tool for the rapid detection and discrimination of S. pneumoniae, S. mitis, and S. oralis from clinical specimens.     

An experimental study on developmental changes of maternal discrimination of infants in crab-eating monkeys (Macaca fascicularis)

Individually reared mother-infant dyads of crab-eating monkeys (Macaca fascicularis) were observed cross-sectionally in their mother-infant relationship. In infants aged from 0 to 5.0 months, rather drastic changes were found both at 0.5-1.0 and 2.0-3.0 months of age. For an explanation of these changes, developmental processes of discrimination between mother and infant were analyzed cross-sectionally by exchanging mother-infant combinations. The results indicated that the first 0.5 months postpartum was characterized as a behaviorally nondiscriminating stage where nipple discrimination by infants was the only exception. The latter half of the first month was the beginning of a nonaggressive discrimination stage by mothers, indicated by lipsmacking and sniffing and in infants by clinging. When infants reached the age of 2.0-3.0 months, the mothers' nonaggressive discrimination with lipsmacking decreased, and her aggressive discrimination of alien infants increased. In addition to maternal visual discrimination of infants' physical appearances, differences in infants' odor and/or their method of nipple contact were suggested to affect the mothers' differential behaviors.     

Transmission of Staphylococcus aureus between mothers and infants in an African setting

Staphylococcus aureus colonization is a risk factor for invasive disease. There is a need to understand S. aureus colonization in infancy as the burden of S. aureus infections in infants is high. We aimed to investigate the transmission of S. aureus between mothers and their newborns during the first year after delivery in an African setting. In a longitudinal cohort study, colonization of Gabonese mother–infant pairs was assessed at delivery and after 1, 9 and 12 months. Swabs were taken from mothers (nares, mammillae) and infants (nares and throat). Isolates were characterized and risk factors for colonization were assessed using a standardized questionnaire. We recruited 311 mothers and 318 infants including seven sets of twins. Maternal and infant colonization rates declined synchronously following a peak after 1 month at 40% (mothers) and 42% (infants). Maternal colonization was a risk factor for S. aureus carriage in infants. Based on spa typing, direct mother-to-infant transmission was evident in 5.6%. Of all methicillin-resistant isolates (<n = 9), 44.4% were related to the USA300 clone; 56.7% (<n = 261) of all S. aureus carried Panton–Valentine leukocidin encoding genes. Direct mother-to-infant transmission was rare and cannot explain the increase of carriage in infants within the first month. A transmission from external sources is likely and challenges the S. aureus infection control in newborns and infants in an African setting. The detection of USA300-related MRSA fuels the concern about the spread of this clone in Central Africa.     

Molecular epidemiology of extended-spectrum beta-lactamases among Escherichia coli isolates collected in a Swedish hospital and its associated health care facilities from 2001 to 2006

The genetic characteristics and molecular epidemiology of extended-spectrum β-lactamases (ESBLs) among Escherichia coli isolates were investigated at a general hospital and its associated health care facilities in Stockholm, Sweden, during the period from 2001 to 2006. Of 87 consecutive nonduplicate ESBL-positive isolates, 80 isolates encoded CTX-M-type ESBLs, 64 of which were group 1 enzymes. TEM-type and OXA-type β-lactamases were encoded in 63 and 59% of the ESBL isolates, respectively. Pulsed-field gel electrophoresis (PFGE) analysis revealed 40 different pulsotypes, consisting of 11 clones accounting for 66% of all isolates, and 29 unique patterns. Moreover, of the 11 clones, clones 1 and 4 comprised half of the clonally related isolates (28 of 57). Clone 1 was a persistent endemic clone in the area throughout the years, and clone 4 emerged in 2003. However, in recent years, clone 1 isolates were no longer predominant and were gradually replaced by new emerging strains. Concerning β-lactamase gene profiles in relation to PFGE pulsotypes, clone-related bla profiles were observed in certain clones, while in most cases different bla profiles could be observed in the same clone, and the same bla profile could be present in different clones. The molecular epidemiology of ESBL-positive E. coli in the area shows shifts in predominant strains and increased clonal diversity over time. The study also indicated that both clonal spread of epidemic strains and transfer of transposable genetic elements might contribute to the proliferation of ESBLs.     

Experience with a flavor in mother's milk modifies the infant's acceptance of flavored cereal.

The present series of studies aimed to investigate whether experience with a flavor in mothers' milk modifies the infants' acceptance of similarly flavored foods at weaning. First, we established, using methods developed in our laboratory, that the ingestion of carrot juice by lactating women produced a sensory change in their milk approximately 2 to 3 hr after the ingestion of the beverage. Second, we randomly formed two groups of breast-fed infants who had been fed cereal for a few weeks but had only experienced cereal prepared with water. Their mothers were asked to consume one of two types of beverages (i.e., carrot juice, water) during the exposure period. Each mother was observed feeding her infant cereal during four test sessions. The first two sessions occurred during the 2 days before the exposure period; in counterbalanced order, infants were fed cereal prepared with water on 1 testing day and cereal prepared with carrot juice on the other. These two test sessions were then repeated following the exposure period. The results demonstrated that the infants who had exposure to the flavor of carrots in their mothers' milk during the exposure period consumed less of the carrot-flavored cereal and spent less time feeding when compared to the control infants whose mothers consumed the water. This may be a form of sensory-specific satiety such that the infants become less responsive to a flavor that they have been extensively exposed to in the very recent past.     

Neisseria meningitidis; clones,carriage, and disease

Neisseria meningitidis, the cause of meningococcal disease, has been the subject of sophisticated molecular epidemiological investigation as a consequence of the significant public health threat posed by this organism. The use of multilocus sequence typing and whole genome sequencing classifies the organism into clonal complexes. Extensive phenotypic, genotypic and epidemiological information is available on the PubMLST website. The human nasopharynx is the sole ecological niche of this species, and carrier isolates show extensive genetic diversity as compared with hyperinvasive lineages. Horizontal gene exchange and recombinant events within the meningococcal genome during residence in the human nasopharynx result in antigenic diversity even within clonal complexes, so that individual clones may express, for example, more than one capsular polysaccharide (serogroup). Successful clones are capable of wide global dissemination, and may be associated with explosive epidemics of invasive disease.     

Relative Importance of Mothers' Versus Medical Staffs' Behavior in the Prediction of Infant Immunization Pain Behavior

Objective: To examine the relative importance of mothers' versusmedical staffs' behavior in the prediction of infant pain duringroutine immunization. Methods: We video-recorded 60 infants' 6- or 18-month immunizations.Recordings were used to code infant pain behavior using theNeonatal Facial Action Coding System (NFCS, R. V. E. Crunau& K. D. Craig, 1987), and maternal and staff vocalizationsusing the Child-Adult Medical Procedure Interaction Scale-Revised(CAMPIS-R, R. L. Blount, J. W. Sturges, & S. W. Powers,1990). Results: Maternal and staff vocalizations showed different patternsof relation with infant pain behavior, depending upon the typeof vocalization. Mothers' distress-promoting behavior (e.g.,reassurance) predicted increased infant pain behavior, whilestaffs' coping-promoting behavior (e.g., distraction) predicteddecreased infant pain behavior. Conclusions: The behavior of medical staff should be an importantconsideration in the assessment of children's medically relatedpain behavior.     

Humoral Immunity to Commensal Oral Bacteria in Human Infants: Salivary Antibodies Reactive with Actinomyces naeslundii Genospecies 1 and 2 during Colonization

The secretory immune response in saliva to colonization by Actinomyces naeslundii genospecies 1 and 2 was studied in 10 human infants from birth to 2 years of age. Actinomyces species were not recovered from the mouths of the infants until approximately 4 months after the eruption of teeth. However, low levels of secretory immunoglobulin A1 (SIgA1) and SIgA2 antibodies reactive with whole cells of A. naeslundii genospecies 1 and 2 were detected within the first month after birth. Although there was a fivefold increase in the concentration of SIgA between birth and age 2 years, there were no differences between the concentrations of SIgA1 and SIgA2 antibodies reactive with A. naeslundii genospecies 1 and 2 over this period. When the concentrations of SIgA1 and SIgA2 antibodies reactive with whole cells of A. naeslundii genospecies 1 and 2 were normalized to the concentrations of SIgA1 and SIgA2 in saliva, the A. naeslundii genospecies 1- and 2-reactive SIgA1 and SIgA2 antibodies showed a significant decrease from birth to 2 years of age. The fine specificities of A. naeslundii genospecies 1- and 2-reactive SIgA1 and SIgA2 antibodies were examined by Western blotting of envelope proteins. Similarities in the molecular masses of proteins recognized by SIgA1 and SIgA2 antibodies, both within and between subjects over time, were examined by cluster analysis and showed considerable variability. Taken overall, our data suggest that among the mechanisms Actinomyces species employ to persist in the oral cavity are the induction of a limited immune response and clonal replacement with strains differing in their antigen profiles.     

A Comprehensive Genetic Study of Streptococcal Immunoglobulin A1 Proteases: Evidence for Recombination within and between Species

An analysis of 13 immunoglobulin A1 (IgA1) protease genes (iga) of strains of Streptococcus pneumoniae, Streptococcus oralis, Streptococcus mitis, and Streptococcus sanguis was carried out to obtain information on the structure, polymorphism, and phylogeny of this specific protease, which enables bacteria to evade functions of the predominant Ig isotype on mucosal surfaces. The analysis included cloning and sequencing of iga genes from S. oralis and S. mitis biovar 1, sequencing of an additional seven iga genes from S. sanguis biovars 1 through 4, and restriction fragment length polymorphism (RFLP) analyses of iga genes of another 10 strains of S. mitis biovar 1 and 6 strains of S. oralis. All 13 genes sequenced had the potential of encoding proteins with molecular masses of approximately 200 kDa containing the sequence motif HEMTH and an E residue 20 amino acids downstream, which are characteristic of Zn metalloproteinases. In addition, all had a typical gram-positive cell wall anchor motif, LPNTG, which, in contrast to such motifs in other known streptococcal and staphylococcal proteins, was located in their N-terminal parts. Repeat structures showing variation in number and sequence were present in all strains and may be of relevance to the immunogenicities of the enzymes. Protease activities in cultures of the streptococcal strains were associated with species of different molecular masses ranging from 130 to 200 kDa, suggesting posttranslational processing possibly as a result of autoproteolysis at post-proline peptide bonds in the N-terminal parts of the molecules. Comparison of deduced amino acid sequences revealed a 94% similarity between S. oralis and S. mitis IgA1 proteases and a 75 to 79% similarity between IgA1 proteases of these species and those of S. pneumoniae and S. sanguis, respectively. Combined with the results of RFLP analyses using different iga gene fragments as probes, the results of nucleotide sequence comparisons provide evidence of horizontal transfer of iga gene sequences among individual strains of S. sanguis as well as among S. mitis and the two species S. pneumoniae and S. oralis. While iga genes of S. sanguis and S. oralis were highly homogeneous, the genes of S. pneumoniae and S. mitis showed extensive polymorphism reflected in different degrees of antigenic diversity.Bacterial immunoglobulin A1 (IgA1) proteases are highly specific endopeptidases that cleave the heavy chain of human IgA1, including its secretory form (S-IgA1), in the hinge region. Such proteases are secreted by a small number of bacteria associated with humans (reviewed in references 20 and 28). IgA1 protease-producing bacteria include the mucosal pathogens Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae, Streptococcus pneumoniae, and Ureaplasma urealyticum as well as some members of resident oral and pharyngeal microfloras. Among the latter are species of Prevotella and Capnocytophaga, Streptococcus sanguis, Streptococcus oralis, and Streptococcus mitis biovar 1.Three different classes of proteinases are represented among the IgA1 proteases, illustrating that cleavage of human IgA1 is a property that has evolved among bacteria through convergent evolution following at least three independent lines. Molecular characterizations have revealed that the IgA1 proteases of Haemophilus and Neisseria are genetically related serine proteinases (1, 25, 29, 30) and that those of S. sanguis and S. pneumoniae are metalloproteinases (8, 32, 40). Studies using specific inhibitors indicate that the IgA1 protease produced by Prevotella melaninogenica is a cysteine proteinase (26). IgA1 proteases are postproline endopeptidases, and those of streptococcal origin cleave one and the same Pro-Thr peptide bond at positions 227 to 228 in the human IgA1 heavy chain (18, 34).The enzymes have been shown to be active in vivo, and by cleaving human IgA1 they interfere with the protective functions of the principal mediator of specific immunity of the upper respiratory tract (reviewed in reference 20). Consequently, these proteases are thought to be important for the ability of the bacteria to colonize human mucosal surfaces in the presence of specific S-IgA1 antibodies. Furthermore, they may constitute an important factor in the pathogenesis of invasive infections (20). Besides, it has been proposed that increased colonization of the pharynges of infants with IgA1 protease-producing bacteria, in particular S. mitis biovar 1, may compromise S-IgA-mediated protection against allergens and lead to atopic sensitization (17).The iga genes encoding IgA1 protease from S. sanguis and S. pneumoniae have been characterized previously (8, 32, 40). Here we report on the cloning and sequencing of iga genes from the other two Streptococcus species, S. oralis and S. mitis, known to produce IgA1-cleaving activity. Furthermore, we have sequenced iga genes from an additional seven strains of S. sanguis representing the four different biovars of this species.     

Phenotypic and Genotypic Changes over Time and across Facilities of Serial Colonizing and Infecting Escherichia coli Isolates Recovered from Injured Service Members

Escherichia coli is the most common colonizing and infecting organism isolated from U.S. service members injured during deployment. Our objective was to evaluate the phenotypic and genotypic changes of infecting and colonizing E. coli organisms over time and across facilities to better understand their transmission patterns. E. coli isolates were collected via surveillance cultures and infection workups from U.S. military personnel injured during deployment (June 2009 to May 2011). The isolates underwent antimicrobial susceptibility testing, pulsed-field gel electrophoresis, and multiplex PCR for phylotyping to determine their resistance profiles and clonality. A total of 343 colonizing and 136 infecting E. coli isolates were analyzed, of which 197 (57%) and 109 (80%) isolates, respectively, produced extended-spectrum β-lactamases (ESBL). Phylogroup A was predominant among both colonizing (38%) and infecting isolates (43%). Although 188 unique pulsed-field types (PFTs) were identified from the colonizing isolates, and 54 PFTs were identified from the infecting isolates, there was a lack of PFT overlap between study years, combat zones, and military treatment facilities. On a per-subject basis, 26% and 32% of the patients with serial colonizing isolates and 10% and 21% with serial infecting isolates acquired changes in their phylogroup and PFT profiles, respectively, over time. The production of ESBL remained high over time and across facilities, with no substantial changes in antimicrobial susceptibilities. Overall, our results demonstrated an array of genotypic and phenotypic differences for the isolates without large clonal clusters; however, the same PFTs were occasionally observed in the colonizing and infecting isolates, suggesting that the source of infections may be endogenous host organisms.     

“Epidemic Clones” of Listeria monocytogenes Are Widespread and Ancient Clonal Groups

The food-borne pathogen Listeria monocytogenes is genetically heterogeneous. Although some clonal groups have been implicated in multiple outbreaks, there is currently no consensus on how “epidemic clones” should be defined. The objectives of this work were to compare the patterns of sequence diversity on two sets of genes that have been widely used to define L. monocytogenes clonal groups: multilocus sequence typing (MLST) and multi-virulence-locus sequence typing (MvLST). Further, we evaluated the diversity within clonal groups by pulsed-field gel electrophoresis (PFGE). Based on 125 isolates of diverse temporal, geographical, and source origins, MLST and MvLST genes (i) had similar patterns of sequence polymorphisms, recombination, and selection, (ii) provided concordant phylogenetic clustering, and (iii) had similar discriminatory power, which was not improved when we combined both data sets. Inclusion of representative strains of previous outbreaks demonstrated the correspondence of epidemic clones with previously recognized MLST clonal complexes. PFGE analysis demonstrated heterogeneity within major clones, most of which were isolated decades before their involvement in outbreaks. We conclude that the “epidemic clone” denominations represent a redundant but largely incomplete nomenclature system for MLST-defined clones, which must be regarded as successful genetic groups that are widely distributed across time and space.