Whole genome sequencing of coagulase positive staphylococci from a dog-and-owner screening survey.

BACKGROUND: Staphylococcus aureus and S. pseudintermedius are the two most common coagulase positive staphylococci (CPS). S. aureus is more prevalent among humans, whereas S. pseudintermedius is more commonly isolated from dogs, however, both can cause various community and hospital acquired diseases in humans. METHODS: In the current study we screened 102 dogs and 84 owners in Hungary. We tested the antibiotic susceptibility of the strains and in order to get a better picture of the clonal relationship of the strains, we used pulsed-field gel electrophoresis. In addition, three pairs of isolates with identical PFGE patterns were whole genome sequenced, MLST and spa types were established. RESULTS: Carriage rate of S. aureus was 23.8% in humans and 4.9% in dogs and two cases of co-carriage were found among dogs and owners. S. pseudintermedius carriage rate was 2.4% and 34.3%, respectively, with only one co-carriage. The isolates were generally rather susceptible to the tested antibiotics, but high tetracycline resistance of S. pseudintermedius strains was noted. The co-carried isolates shared almost the same resistance genes (including tet(K), bla(Z), norA, mepR, lmrS, fosB) and virulence gene pattern. Apart from the common staphylococcal enzymes and cytotoxins, we found enterotoxins and exfoliative toxins as well. The two S. aureus pairs belonged to ST45-t630, ST45-t671 and ST15-t084, ST15-t084, respectively. The co-carried S. pseudintermedius isolates shared the same housekeeping gene alleles determining a novel sequence type ST1685. CONCLUSIONS: Based on the genomic data, dog-owner co-carried strains displayed only insignificant differences therefore provided evidence for potential human-to-dog and dog-to-human transmission.

Determining the serotype composition of mixed samples of pneumococcus using whole-genome sequencing.

Serotyping of Streptococcus pneumoniae is a critical tool in the surveillance of the pathogen and in the development and evaluation of vaccines. Whole-genome DNA sequencing and analysis is becoming increasingly common and is an effective method for pneumococcal serotype identification of pure isolates. However, because of the complexities of the pneumococcal capsular loci, current analysis software requires samples to be pure (or nearly pure) and only contain a single pneumococcal serotype. We introduce a new software tool called SeroCall, which can identify and quantitate the serotypes present in samples, even when several serotypes are present. The sample preparation, library preparation and sequencing follow standard laboratory protocols. The software runs as fast as or faster than existing identification tools on typical computing servers and is freely available under an open source licence at https://github.com/knightjimr/serocall. Using samples with known concentrations of different serotypes as well as blinded samples, we were able to accurately quantify the abundance of different serotypes of pneumococcus in mixed cultures, with 100 % accuracy for detecting the major serotype and up to 86 % accuracy for detecting minor serotypes. We were also able to track changes in serotype frequency over time in an experimental setting. This approach could be applied in both epidemiological field studies of pneumococcal colonization and experimental laboratory studies, and could provide a cheaper and more efficient method for serotyping than alternative approaches.

Evaluating post-vaccine expansion patterns of pneumococcal serotypes.

BACKGROUND: Streptococcus pneumoniae remains a leading cause of morbidity and mortality. Pneumococcal conjugate vaccines (PCVs) are effective but target only a fraction of the more than 90 pneumococcal serotypes. As a result, the introduction of PCVs has been followed by the emergence of non-vaccine serotypes. With higher-valency PCVs currently under development, there is a need to understand and predict patterns of serotype replacement to anticipate future changes. METHODS: In this study, we fit a hierarchical Bayesian regression model to evaluate patterns of change in serotype prevalence post-PCV introduction in Israel from 2009 to 2016. RESULTS: We found that the assumption that non-vaccine serotypes increase by the same proportion overestimates changes in serotype prevalence in Jewish and Bedouin children. Furthermore, pre-vaccine prevalence was positively associated with increases in prevalence over the study period. From our analyses, serotypes 12F, 8, 16F, 33F, 9N, 7B, 10A, 22F, 24F, and 17F were estimated to have gained the most cases of invasive pneumococcal disease through serotype replacement in the Jewish population. However, this model also failed to quantify some additional cases gained, suggesting that changes in carriage in children alone may be insufficient to explain serotype replacement in disease. CONCLUSIONS: Understanding of serotype replacement is important as higher-valency vaccines are introduced.

Co-carriage of Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis among three different age categories of children in Hungary.

BACKGROUND: The nasopharynx can from time to time accommodate otherwise pathogenic bacteria. This phenomenon is called asymptomatic carriage. However, in case of decreased immunity, viral infection or any other enhancing factors, severe disease can develop. Our aim in this study was to survey the nasal carriage rates of four important respiratory pathogens in three different age groups of children attending nurseries, day-care centres and primary schools. This is the first study from Hungary about the asymptomatic carriage of H. influenzae and M. catarrhalis. METHODS: Altogether 580 asymptomatic children were screened in three Hungarian cities. Samples were collected from both nostrils with cotton swabs. The identification was based on both colony morphology and species-specific PCRs. Serotyping was performed for S. pneumoniae, H. influenzae and M. catarrhalis. Antibiotic susceptibility was determined with agar dilution, according to the EUCAST guidelines. Clonality was examined by PFGE. RESULTS AND CONCLUSIONS: Whereas the carriage rates of S. pneumoniae, H. influenzae and M. catarrhalis clearly decreased with age, that of S. aureus showed an opposite tendency. Multiple carriage was least prevalent if S. aureus was one of the participants. The negative association between this bacterium and the others was statistically significant. For pneumococcus, the overall carriage rate was lower compared to earlier years, and PCV13 serotypes were present in only 6.2% of the children. The majority of H. influenzae isolates was non-typeable and no type b was detected; serotype A was dominant among M. catarrhalis. All four bacteria were more sensitive to antibiotics compared to clinical isolates. No MRSAs were detected, but we found three mupirocin resistant strains. The positive effect of Hib- and PCV-vaccination is undoubted. Continued surveillance of these pathogens is required.

Variation of growth characteristics of pneumococcus with environmental conditions.

BACKGROUND: Pneumococcus is exposed to a variety of temperature and oxygen levels in the upper respiratory tract and as it invades the lung, tissues, and blood. We sought to determine the effect of environmental variability on growth in vitro and to assess variability between strains. We evaluated the effect of temperature and oxygen on the growth of 256 isolates representing 53 serotypes, recovered from healthy carriers and disease patients. Strains were grown at a range of temperatures, anaerobically or in ambient air with catalase, and were monitored by reading the optical density. Regression models evaluated variation in the characteristics of the growth curves. RESULTS: Most isolates grew to the maximal density at low temperatures (~33C) and under aerobic conditions. There was considerable variability between strains, and some of this variability was linked to serotype. However, capsule-switch experiments suggest that the production of different capsules might not be sufficient to explain this variation, suggesting there could be interactions between the capsule and genetic background. CONCLUSIONS: Pneumococcal strains vary in how they respond to environmental variations, some of this variation can be explained by the capsule type being produced, but capsule production itself is not sufficient to explain the variability. This variability could help to explain why different lineages of pneumococcus are more common in carriage or disease.

High prevalence of group B streptococcus ST17 hypervirulent clone among non-pregnant patients from a Hungarian venereology clinic.

BACKGROUND: Although Streptococcus agalactiae is the leading causative agent of neonatal sepsis and meningitis, recently it is increasingly isolated from non-pregnant adults. The relation between its presence in the genitourinary tract and manifested clinical symptoms of STD patients remains an open question. In this study, a complex epidemiological investigation of GBS isolates from a venerology clinic was performed. METHODS: Ninety-six GBS isolates were serotyped and their genetic relatedness determined by PFGE. MLST was also performed for a subset of 20 isolates. The antibiotic susceptibility was tested with agar dilution. Surface proteins and the ST-17 hypervirulent clone was detected by PCR. RESULTS: The serotype prevalence was the following: V (29.2%), III (27.1%), Ia (22.9%), IV (10.4%), II (5.2%) and Ib (4.2%). A strong association was demonstrated between surface protein genes and serotypes. All isolates were fully susceptible to penicillin, but erythromycin and clindamycin resistance was high (41.7 and 35.4%, respectively), and 8 phenotypically macrolide sensitive isolates carried the ermB gene. 21.9% of all strains belonged to the hypervirulent ST17 clone, most being of serotype III and all were rib +. We found a few serotype IV isolates belonging to several STs and one serotype V/ST110 strain, containing a 44-bp deletion in the atr allele. CONCLUSIONS: The presence of silent ermB genes is of worry, as their expression upon macrolide exposure could lead to unforeseen therapeutic failure, while clindamycin is used for intrapartum antibiotic prophylaxis, in case of penicillin allergy. The other alarming result is the high prevalence of ST17 among these strains from STD patients, who could be sources of further infections. This is the first report from Hungary providing both serotyping and genotyping data of GBS isolates. These results could be helpful for vaccine production as the major vaccine candidates are capsular antigens or surface proteins.

Vaccine-driven serotype-rearrangement is seen with latency in clinical isolates: Comparison of carried and clinical pneumococcal isolates from the same time period in Hungary.

Young children - the main asymptomatic carriers of pneumococcus - are often the source of pneumococcal infections. PCV13 replaced PCV7 in 2010 in Hungary and it became a mandatory vaccine in 2014. In this work we surveyed the effect of vaccination in three groups: in healthy children under 7 years; in children of the same age but infected with pneumococcus (P1); in older patients (P2) who were very likely not vaccinated. Nasal swabs were taken from 522 healthy children to screen pneumococcal carriage between March 2015 and May 2016. In the same time period, 146 clinical isolates were collected, mainly from mucosal infections. Serotypes, antibiotic susceptibility and clonality of the isolates was determined and compared. The carriage rate was 39.1%. Regarding carriage, the serotype distribution showed the total disappearance of serotypes 3 and 6A compared to former Hungarian studies. The prevalence of PCV13 serotypes was only 5.8% represented by three serotypes (19F, 19A, 9V). Of note, serotype 19A (a very resistant and invasive type) also decreased significantly. In the patient groups, PCV13 prevalence was higher: 17.5% (P1) and 32.6% (P2). Although serotype 3 was present in P1 (7.9%), the leading serotype was 23B (22.2%), a non-vaccine type (NVT). P2 showed the most diverse serotype distribution, but serotype 3 was predominant here (15.7%). Pneumococcal isolates from the patients were more resistant towards the tested antibiotics compared to those from carriers. PCV13 seems to be highly successful in reducing the prevalence of vaccine serotypes. The serotype-rearrangement can be seen also among clinical isolates, albeit somewhat later in time. Fortunately, the replacing serotypes are less invasive and less resistant, but, most worrisome, serotype 19F can be found again with increased frequency among carriage isolates and mucosal infections. Further surveillance is needed to carefully monitor such successful, antibiotic resistant "refugees".

Bicarbonate Inhibits Bacterial Growth and Biofilm Formation of Prevalent Cystic Fibrosis Pathogens.

We investigated the effects of bicarbonate on the growth of several different bacteria as well as its effects on biofilm formation and intracellular cAMP concentration in Pseudomonas aeruginosa. Biofilm formation was examined in 96-well plates, with or without bicarbonate. The cAMP production of bacteria was measured by a commercial assay kit. We found that NaHCO3 (100 mmol l-1) significantly inhibited, whereas NaCl (100 mmol l-1) did not influence the growth of planktonic bacteria. MIC and MBC measurements indicated that the effect of HCO 3 - is bacteriostatic rather than bactericidal. Moreover, NaHCO3 prevented biofilm formation as a function of concentration. Bicarbonate and alkalinization of external pH induced a significant increase in intracellular cAMP levels. In conclusion, HCO 3 - impedes the planktonic growth of different bacteria and impedes biofilm formation by P. aeruginosa that is associated with increased intracellular cAMP production. These findings suggest that aerosol inhalation therapy with HCO 3 - solutions may help improve respiratory hygiene in patients with cystic fibrosis and possibly other chronically infected lung diseases.

High prevalence of Staphylococcus aureus nasal carriage among children in Szolnok, Hungary.

We collected nasal samples from 1,390 healthy 3-7 years old children in Szolnok city, Hungary, in 2012. We detected 476 Staphylococcus aureus isolates from 474 children. In two occasions, two different S. aureus were isolated, based on hemolysis type and pulsed-field gel electrophoresis pattern. S. aureus carriage rate was calculated to be 34.1% similar to others studies. Male gender was found to be a risk factor for carriage by statistical analysis. Altogether, four methicillin-resistant S. aureus (MRSA) strains were detected by mecA polymerase chain reaction, which means 0.8% community-acquired MRSA prevalence among the S. aureus isolates. All MRSA strains harbored the SCCmec type IV cassette (typical for CA-MRSA) and belonged to ST45 by multilocus sequence typing. During antibiotic susceptibility testing, we measured the following resistance rates: 0.0% for mupirocin, 0.2% for ciprofloxacin, 0.6% for gentamicin and oxacillin, 3.4% for tetracycline, 9.5% for clindamycin, 10.3% for erythromycin, and 91.4% for penicillin, which are generally lower compared with Hungarian clinical isolates.

First description of a catalase-negative Staphylococcus aureus from a healthy carrier, with a novel nonsense mutation in the katA gene.

We have screened 2568 healthy individuals (mostly children) for Staphylococcus aureus and Streptococcus pneumoniae nasal carriage between 2010 and 2012. Out of the isolated 751 S. aureus strains, we found one methicillin-sensitive catalase-negative S. aureus (CNSA). Our CNSA isolate possessed a novel nonsense point mutation in the katA gene leading to early truncation of the protein product. The strain was resistant to penicillin and erythromycin, but sensitive to all other tested antibiotics and carried the enterotoxin A gene. It belonged to sequence type 5 (ST5), which is a successful, worldwide spread, usually MRSA clone. Catalase has been described as a virulence factor strictly required for nasal colonisation, and this is the first case contradicting this theory, as all previous CNSA isolates derived from infections. This is the first report of a CNSA from a symptomless carrier as well as the first occurrence in Hungary.

Epidemiology and antibiotic sensitivity of Staphylococcus aureus nasal carriage in children in Hungary.

The aim of this study was to assess the Staphylococcus aureus nasal carriage rate in healthy children all over Hungary and to specify some risk factors, the antibiotic resistance patterns of the bacteria, and their genetic relatedness. In total, 878 children (aged 3-6 years) were screened at 21 day-care centers in 16 different cities in Hungary, between February 2009 and December 2011. Samples taken from both nostrils were cultured on blood agar, and suspected S. aureus isolates were identified by ß-hemolysis, catalase positivity, clump test, and nucA PCR. Methicillin-resistant strains were screened by mecA and mecC PCR. Antibiotic susceptibility was determined by agar dilution or gradient test strips. Pulsed-field gel electrophoresis was used for genotyping. S. aureus carriage rate was found to be 21.3%, which correlates well with international data. We found no statistically significant correlation between the gender or the sibling status and S. aureus carriage. All isolates were sensitive to oxacillin, trimethoprim-sulfamethoxazole, and mupirocin. The resistance rates for erythromycin, ciprofloxacin, clindamycin, gentamicin, and tetracycline were 7.5%, 0.5%, 1.1%, 3.7%, and 4.3%, respectively. The isolates showed very high genetic diversity. In summary, carried S. aureus isolates are more sensitive to antibiotics compared with clinical isolates in Hungary, and methicillin-resistant S. aureus carriage rate is very low yet.

Radical serotype rearrangement of carried pneumococci in the first 3 years after intensive vaccination started in Hungary.

UNLABELLED: Streptococcus pneumoniae is responsible for a significant amount of morbidity and mortality worldwide. Healthy carriers, mainly young children, are the most important sources of infections. In the current study, we aimed to determine the changes that have occurred since the introduction of PCV-7 in Hungary. Nasal specimens were collected from 1,022 healthy children aged 3-6 years attending day-care centres. After thorough identification, pneumococcal isolates were serotyped, and their antibiotic sensitivity was determined. The carriage rate was found to be 34.9%. A huge serotype rearrangement was detected compared to earlier results, with the previously leading serotype 14 having completely disappeared. Serotypes 11A, 35F, 19A, 6B, 15B, 3 and 38 were most prevalent, and 29 different types were identified in total. The PCV-7 types were responsible for 16.5% of all serotypes, and 36.0% are not covered by any pneumococcal vaccines. The isolates were sensitive to most tested antibiotics, except erythromycin (resistance was 21.6%). Only one penicillin-resistant strain was found. The newly and rapidly emerging non-vaccine serotypes are much more sensitive, except serotype 19A. CONCLUSION: Due to PCV vaccination, a complete serotype arrangement occurred also in Hungary. The old "paediatric" serotypes were replaced by serotypes 11A, 35F, 19A, 6B, 15B, 3 and 38.

Changes in the serotypes of Hungarian pneumococci isolated mainly from invasive infections: a review of all available data between 1988 and 2011.

Streptococcus pneumoniae is responsible for a high level of morbidity and mortality, especially among children. For a long time, only the polysaccharide vaccine was available against pneumococcal infections, but in the last decade special conjugate vaccines were developed for paediatric use. These vaccines have made a deep impact on serotype distribution all over the world, by suppressing those serotypes included in the vaccines, while new, previously rare types emerged. These changes have been monitored closely in numerous publications all over the world. Nevertheless, data on pneumococcal serotypes in Hungary were mostly published in Hungarian, therefore not available in the international literature. In this meta-analysis, our aim was to collect and summarise all available data, and try to follow the changes observed after the introduction of the conjugate vaccines.

[Drastic changes in serotypes of carried pneumococci due to an increased vaccination rate in Hungary]. / A hordozott pneumococcusok szerotípusainak drasztikus változása a megnövekedett átoltottság hatására hazánkban.

UNLABELLED: Introduction of the conjugate pneumococcal vaccine into the voluntary childhood vaccine program in Hungary in April 2009 resulted in a sharp increase of the vaccination rate. However, changes in serotypes as a consequence of vaccination should be considered. AIMS: The aim of the authors was to compare pneumococci isolated from children with high-level and low-level vaccination rates. METHODS: Nasal specimens from 854 children attending 20 nurseries at various locations in Hungary have been collected since 2009. The serotypes, antibiotic susceptibility and genetic relatedness of the isolated pneumococci were determined. RESULTS: 324 strains were isolated, and the carriage rate was 37.94%. The strains were sensitive to most antibiotics, except for macrolides. A definite suppression of vaccine types was detected during these 3 years, from the initial 78.85% to 35.30%. CONCLUSIONS: The authors conclude that the results reflect the efficacy of the vaccine, which underlines the need for the inclusion of pneumococcal vaccine into the list of obligatory vaccines.

Nasal carriage of Streptococcus pneumoniae among Hungarian children before the wide use of the conjugate vaccine.

Streptococcus pneumoniae is responsible for a significant amount of morbidity and mortality worldwide, especially among children <5 years. Healthy carriers are the most important sources of infections and the carriage also peaks in the first years of life, especially among children attending communities. In this study, for the first time in Hungary, we surveyed the nasal carriage of healthy children, just before the use of the conjugate vaccine started increasing.Nasal specimens of 358 children were cultured and pneumococci isolated. The strains were serotyped with antisera and PCR, genotyped by PFGE and their antibiotic sensitivity determined by agar dilution method.The carriage rate was 37.71%. The isolates were sensitive to most tested antibiotics, except for macrolides. In this cohort of specimens still the widespread, so-called "pediatric serotypes" dominated (14, 19F, 23F, 6A, 6B in ranking order), but three of the previously rare types: 15B, 11A and 13 were represented already by 21.5% of all strains and also a few other rare non-vaccine types (e.g. 10A or 37) were detected.The calculated vaccine coverage was 55.6% for PCV-7, 69.6% for PCV-13 and 86.7% for Pneumovax. In this cohort, only 15.9% of the children (n = 57) were vaccinated. The carriage rate of PCV-7 vaccinated children was significantly lower (30.4%) than that of the non-vaccinated group (39.2%). The clonality of the isolates was significant within each group, revealing the extensive bacterium exchange among children.

A marked shift in the serotypes of pneumococci isolated from healthy children in Szeged, Hungary, over a 6-year period.

Streptococcus pneumoniae is an important pathogen with significant morbidity and mortality rates worldwide, especially among children <5 years. Healthy carriers are the most important sources of pneumococcal infections, and the nasopharyngeal colonisation is the most prevalent among children attending communities such as day-care centres (DCCs). The conjugate pneumococcal vaccines (PCVs) were shown to have an impact on the colonisation, and so play an important role in inhibiting infections. In this study we compared the nasal carriage of healthy children attending DCCs in Szeged, Hungary in 2003/2004, when nobody was vaccinated, and in 2010, when already 1/5 of the children received PCV-7. Significant differences were observed in the serotype distribution, representing a marked shift from the previously widespread vaccine-types (mostly 6A or 14) to others (11A and 23F). The new serotypes showed higher antibiotic susceptibility. The bacterium exchange between children was clear from the pulsed-field gel electrophoresis (PFGE) patterns, and the circulation of certain international clones plays also a role in these dynamic changes.