Molecular characterization of Vibrio cholerae O1 isolates obtained from outbreaks in the Philippines, 2015–2016

Introduction. The Philippines, comprising three island groups, namely, Luzon, Visayas and Mindanao, experienced an increase in cholera outbreaks in 2016. Previous studies have shown that Vibrio cholerae isolates obtained from the Philippines are novel hybrid El Tor strains that have evolved in the country and are clearly distinct from those found in Mozambique and Cameroon. Gap statement. The characterization of the strains isolated from outbreaks has been limited to phenotypic characteristics, such as biochemical and serological characteristics, in most previous studies. Aim. We performed multilocus variable-number tandem repeat (VNTR) analysis (MLVA) for V. cholerae isolates obtained from 2015 to 2016 to further characterize and understand the emergence and dissemination of the strains in the Philippines. Methodology. A total of 139 V . cholerae O1 Ogawa biotype El Tor isolates were obtained from the Philippines during diarrhoeal outbreaks in 18 provinces between 2015 and 2016. VNTR data were analysed to classify the MLVA profiles where the large-chromosome types (LCTs) were applied for grouping. Results. We identified 50 MLVA types among 139 isolates originating from 18 provinces, and 14 LCTs. The distribution of the LCTs was variable, and a few were located in specific areas or even in specific provinces. Based on eBURST analysis, 99 isolates with 7 LCTs and 32 MLVA types belonged to 1 group, suggesting that they were related to each other. LCT A was predominant (n=67) and was isolated from Luzon and Visayas. LCT A had 14 MLVA types; however, it mostly emerged during a single quarter of a year. Eight clusters were identified, each of which involved specific MLVA type(s). The largest cluster involved 23 isolates showing 3 MLVA types, 21 of which were MLVA type A-14 isolated from Negros Occidental during quarter 4 of 2016. Comparative analysis showed that almost all isolates from the Philippines were distinct from those in other countries. Conclusions. The genotypic relationship of the V. cholerae isolates obtained during outbreaks in the Philippines was studied, and their emergence and dissemination were elucidated. MLVA revealed the short-term dynamics of V. cholerae genotypes in the Philippines.

Spread of clonal genovar E Chlamydia trachomatis among men who have sex with men

In a previous study, we developed a Multi-Locus VNTRs Analysis (MLVA) typing system, called MLVA-5, for the discrimination of Chlamydia trachomatis genovar E strain. The results suggested the clonal spread of a MLVA-5 type 21 strain among men who have sex with men (MSM). We applied the MLVA-5 typing method on 157 French anorectal genovar E specimens and 19 Swedish specimens collected between 2010 and 2015. A total of 29 MLVA-5 types was obtained, with three predominant types among French samples: 78 specimens belonged to MLVA-5 type 21, two other types, 11 and 13, included 9 and 14 specimens, respectively. In 15 cases, one unique MLVA-5 type was observed for a single patient, 7 of which were new types not previously described. The distribution of MLVA-5 types according to sexual orientation showed that the 7 anorectal specimens from heterosexual patients belonged to 6 genotypes, and the 12 anorectal specimens from bisexual patients comprised eight types. The 95 anorectal specimens from MSM were distributed into 22 types, but 55 (57.9%) of them belonged to MLVA-5 type 21. Among the Swedish specimens from MSM, eight were from MLVA-type 21 (4 urines and 4 anorectal specimens). The results support the hypothesis of the spread of clonal genovar E strain among MSM.

Macrolide Resistance, Clinical Features, and Cytokine Profiles in Taiwanese Children with Mycoplasma pneumoniae Infection

Background The factors to predict the progression of Mycoplasma pneumoniae infection remain inconclusive. Therefore, we investigated macrolide resistance prevalence, M. pneumoniae genotype, and clinical characteristics of childhood M. pneumoniae respiratory tract infections in Taiwan. Methods A total of 295 children hospitalized with respiratory tract infections with positive serological M. pneumoniae immunoglobulin M test results were enrolled in this 3-year prospective study. Oropharyngeal swabs were obtained for M. pneumoniae cultures and PCR tests. All M. pneumoniae specimens were further characterized by P1 typing, multilocus variable-number tandem-repeat analysis (MLVA), and macrolide resistance genotyping. The clinical characteristics and blood cytokine profiles were analyzed accordingly. Results Of 138 M. pneumoniae specimens, type I P1 was the predominant (136/138, 98.6%). MLVA type P (4-4-5-7-2) was the leading strain (42/138, 30.4%), followed by type J, U, A, and X. The overall macrolide-resistant rate was 38.4% (53/138); the resistance rate increased dramatically yearly: 10.6% in 2017, 47.5% in 2018, and 62.5% in 2019 (P < .001). All macrolide-resistant M. pneumoniae (MRMP) harbored the A2063G mutation and were MLVA type 4-5-7-2 (49/53, 92.5%), especially type U and X. No significant differences in clinical symptoms, duration of hospital stay, and radiographic findings were identified among patients between MRMP and macrolide-sensitive M. pneumoniae (MSMP) groups. Patients with MRMP infection had more febrile days before and during hospitalization; higher IL-13 and IL-33 levels than patients with MSMP infection (P < .005). Conclusions MRMP surged in Taiwan throughout the study period, but macrolide resistance was not a determinant factor of clinical severity.

Methicillin-Resistant Staphylococcus aureus Prevalence Among Healthcare Workers in Contact Tracings in a Dutch Hospital

Background: In The Netherlands, the national guidelines on Methicillin-Resistant Staphylococcus aureus (MRSA) prevention and control advocate screening of healthcare workers (HCWs) after unprotected exposure to MRSA carriers. Although this strategy is largely successful, contact tracing of staff is a time-consuming and costly component. We evaluated our contact tracing policy for HCWs over the years 2010–2018. Methods: A retrospective, observational study was performed in a Dutch teaching hospital. All HCWs who had unprotected contact with an MRSA carrier were included in contact tracing. When there had been a long period of unprotected admission prior to an MRSA finding, or when the index case was an HCW, the entire (nursing) team was tested. All samples of HCWs who were tested for MRSA carriage as part of contact tracing from 2010 until 2018 were included. A pooled nose, throat, and perineum swab was collected using the eSwab medium (Copan) and inoculated on chromID MRSA agar plates (bioMérieux) after enrichment in a broth. Molecular typing was performed using multiple-locus variable number of tandem repeat analysis (MLVA). Results: In total, we included 8,849 samples (range, 677–1,448 samples per year) from 287 contact tracings (range, 26–55 contact tracings per year). Overall, 32 HCWs were colonized with MRSA (0.36%; 95% CI, 0.26%–0.51%). None of them developed a clinical infection. Moreover, 8 HCWs (0.10%; 95% CI, 0.05%–0.19%) were colonized with the same MLVA type as the index case and were detected in 6 of 287 contact tracings (2%). In 4 of 8 of these cases, a positive HCW was the index for undertaking contact tracing. In 3 of 8 cases, it was clear that the HCW who was identified in the contact tracing was the source of the outbreak and was the cause of invasive MRSA infections in patients. Notably, a different MLVA type as the index case was found in 24 HCWs (0.27%; 95% CI, 0.18%–0.40%) of whom 7 of 24 HCWs (29.2%) were intermittent carriers. Conclusions: This study revealed a sustained low MRSA prevalence among samples in contact tracing of HCWs over 9 years. Furthermore, it shows that when MRSA contact tracing is performed according to the national guideline, only 1 of 1,000 samples results in a secondary case. This is similar to the population carriage rate of MRSA in The Netherlands. More frequently, an unrelated strain is found. These findings raise questions regarding the efficacy of the current strategy to perform contact tracing after unprotected exposure.Funding: NoneDisclosures: None

865. Methicillin-resistant Staphylococcus aureus (MRSA) prevalence among healthcare workers (HCW) in contact tracings in a Dutch teaching hospital, 2010-2018

Background In The Netherlands, the national guidelines on MRSA prevention and control advocate screening of HCW after unprotected exposure to MRSA carriers. Although this strategy at large is successful, contact tracing of staff is a time consuming and costly component. We evaluated our contact tracing policy for HCW over the years 2010 – 2018. MRSA prevalence among samples in contact tracing Methods A retrospective, observational study was performed in a Dutch teaching hospital. All HCW who had unprotected contact with an MRSA carrier were included in contact tracing. When there had been a long period of unprotected admission prior to an MRSA finding, or when the index case was a HCW, than the entire (nursing) team was tested. All samples of HCWs who were tested for MRSA carriage as part of contact tracing from 2010 until 2018 were included. A pooled nose, throat and perineum swab was collected using the eSwab medium (Copan) and inoculated on chromID MRSA agar plates (bioMérieux) after enrichment in a broth. Results In total, we included 8,849 samples (range: 677 – 1,448 samples per year) from a total of 287 contact tracings (range: 26 – 55 contact tracings per year). Thirty two HCWs were colonized with MRSA (0.36%; 95%CI 0.26 – 0.51). None of them developed a clinical infection. Eight HCWs (0.10%; 95%CI 0.05% – 0.19%) were colonized with the same MLVA type as the index case, and were detected in 6/287 contact tracings (2%). In 4/8 of these cases, a positive HCW was the index for undertaking contact tracing. In 3/8 cases it was clear that the HCW who was identified in the contact tracing was the source of the outbreak and was the cause of invasive MRSA infections in patients. Notably, a different MLVA type as the index case was found in 24 HCWs (0,27%; 95%CI 0,18 – 0,40) of which 7/24 HCW (29,2%) were intermittent carriers. Conclusion This study revealed a sustained low MRSA prevalence among samples in contact tracing of healthcare workers, over nine years. Furthermore, it shows that when MRSA contact tracing is performed according to the national guideline only 1 out 1000 samples results in a secondary case. This is similar to the population carriage rate of MRSA in The Netherlands. More frequently, an unrelated strain is found. These findings raise question marks regarding the efficacy of the current strategy to perform contact tracing after unprotected exposure. Disclosures All Authors: No reported disclosures

Molecular Characterization of Mycoplasma pneumoniae Isolates in the United States from 2012 to 2018

ABSTRACT Mycoplasma pneumoniae is a major cause of community-acquired pneumonia. There are limited data in the United States on the molecular epidemiological characteristics of M. pneumoniae. We collected 446 M. pneumoniae-positive specimens from 9 states between August 2012 and October 2018. Culture, antimicrobial susceptibility testing, P1 subtyping, and multilocus VNTR (variable-number tandem repeats) analysis (MLVA) were performed to characterize the isolates. Macrolide-resistant M. pneumoniae (MRMp) was detected in 37 (8.3%) specimens. P1 subtype 2 (P1-2) was the predominant P1 subtype (59.8%). P1 subtype distribution did not change significantly chronologically or geographically. The macrolide resistance rate in P1 subtype 1 (P1-1) samples was significantly higher than that in P1-2 (12.9% versus 5.5%). Six P1-2 variants were identified, including two novel types, and variant 2c was predominant (64.6%). P1-2 variants were distributed significantly differently among geographic regions. Classical P1-2 was more frequent in lower respiratory tract specimens and had longer p1 trinucleotide repeats. Classical P1-2 was most common in MRMp (35.7%), while variant 2c was most common in macrolide-susceptible M. pneumoniae (67.5%). Fifteen MLVA types were identified; 3-5-6-2 (41.7%), 4-5-7-2 (35.3%), and 3-6-6-2 (16.6%) were the major types, and four MLVA clusters were delineated. The distribution of MLVA types varied significantly over time and geographic location. The predominant MLVA type switched from 4-5-7-2 to 3-5-6-2 in 2015. MLVA type was associated with P1 subtypes and P1-2 variant types but not with macrolide resistance. To investigate the M. pneumoniae genotype shift and its impact on clinical presentations, additional surveillance programs targeting more diverse populations and prolonged sampling times are required.

Prolonged carriage of (livestock-associated) MRSA in individuals without professional livestock contact

Objectives To investigate prolonged carriage of MRSA in adults from the general population living in a livestock-dense area, using WGS. Methods A cross-sectional study during 2014–15 among 2492 adults without professional livestock contact identified 14 (0.6%) nasal MRSA carriers, 10 of which carried livestock-associated (LA)-MRSA of multiple-locus variable-number tandem repeat analysis (MLVA) complex (MC) 398. Two years later, 12 MRSA-positive and 88 MRSA-negative participants provided a second nasal swab and filled in a short questionnaire. Isolates from persons who were MRSA positive at both timepoints were compared using MLVA and isolates with the same MLVA type were sequenced. The WGS data were used for core-genome MLST (cgMLST) and resistome analysis, including sequenced isolates from the national MRSA surveillance. Results All MRSA-negative persons tested negative again, while 6 of the 12 initially MRSA-positive persons tested positive again. MLVA revealed that isolate pairs from five individuals had the same MLVA type, of which three were LA-MRSA. cgMLST showed that the distance between these isolate pairs ranged between 3 and 13 genes, while the minimum distance to unrelated isolates from the national MRSA surveillance was 38 genes. Moreover, the resistome present in the five isolate pairs was identical within each pair. None of the prolonged carriers was hospitalized during the 3 months before the sampling moment and none of them with LA-MRSA had contact with livestock in this period. Conclusions Prolonged carriage of MRSA, including LA-MRSA, can be demonstrated after more than 30 months in persons without professional livestock contact.

In vivo passage of Salmonella Typhimurium results in minor mutations in the bacterial genome and increases in vitro invasiveness

Eggs and raw or undercooked egg-containing food items are frequently identified as the bacterial source during epidemiolocal investigation of Salmonella outbreaks. Multi-locus variable number of tandem repeats analysis (MLVA) is a widely used Salmonella typing method enabling the study of diversity within populations of the same serotype. In vivo passage, however, has been linked with changes in MLVA type and more broadly the Salmonella genome. We sought to investigate whether in vivo passage through layer hens had an effect on MLVA type as well as the bacterial genome and whether any mutations affected bacterial virulence. Layer hens were infected with either Salmonella Typhimurium DT9 (03-24-11-11-523) as part of a single infection or were co-infected with an equal amount of Salmonella Mbandaka. Salmonella shedding in both single and co-infected birds was variable over the course of the 16-week experiment. Salmonella Typhimurium and Salmonella Mbandaka were identified in feces of co-infected birds. Salmonella colonies isolated from fecal samples were subtyped using MLVA. A single change in SSTR-6 was observed in Salmonella Typhimurium strains isolated from co-infected birds. Isolates of Salmonella Typhimurium of both the parent (03-24-11-11-523) and modified (03-24-12-11-523) MLVA type were sequenced and compared with the genome of the parent strain. Sequence analysis revealed that in vivo passaging resulted in minor mutation events. Passaged isolates exhibited significantly higher invasiveness in cultured human intestinal epithelial cells than the parent strain. The microevolution observed in this study suggests that changes in MLVA may arise more commonly and may have clinical significance.

Επιδημιολογική και εργαστηριακή διερεύνηση του κοκκύτη στα παιδιά

ΠΕΡΙΛΗΨΗ Ο κοκκύτης είναι μια εξαιρετικά μεταδοτική λοίμωξη του αναπνευστικού, η οποία οφείλεται κυρίως στην B. pertussis. Παρά τη μακροχρόνια εφαρμογή προγραμμάτων εμβολιασμού στις αναπτυγμένες χώρες, η λοίμωξη από B. pertussis έχει αναζωπυρωθεί από τις αρχές του 21ου αιώνα. Πιθανά αίτια για την εν λόγω επανεμφάνιση αποτελούν η βελτίωση της επιτήρησης και της διάγνωσης της λοίμωξης, η φθίνουσα ανοσία, η ποικίλη αποτελεσματικότητα των ακυτταρικών εμβολίων, αλλά και η προσαρμογή του βακτηριδίου στην επαγόμενη από το εμβόλιο ανοσία. Σκοπός: Σκοπός της μελέτης ήταν η διερεύνηση της επιδημιολογικής συμπεριφοράς του κοκκύτη καθώς και η μοριακή τυποποίηση της B. pertussis με σύγχρονες τεχνικές στην πενταετία 2010 - 2015, χρονική περίοδο όπου ο εμβολιασμός στη χώρα μας για τη νόσο ήταν υποχρεωτικός και η εμβολιαστική κάλυψη υψηλή. Ασθενείς - Μέθοδοι: Η διάγνωση και η μοριακή τυποποίηση της B. pertussis έγινε σε ρινοφαρυγγικά δείγματα παιδιών, που νοσηλεύτηκαν στο Γενικό Νοσοκομείο Παίδων ‘Η Αγία Σοφία’ κατά την περίοδο 2010 - 2015. Με την real - time Polymerase Chain Reaction (PCR) ανιχνεύθηκαν οι DNA στόχοι IS481 / IS1001 / recA των B. pertussis / parapertussis / holmesii αντίστοιχα. Ο πολυμορφισμός της B. pertussis μελετήθηκε με multi - locus variable number tandem repeat analysis (MLVA) και με sequence - based typing της toxin promotor region (ptP) σε DNA θετικών δειγμάτων. Αποτελέσματα: Από τα 1150 δείγματα στα οποία διενεργήθηκε PCR, 300 ήταν θετικά για B. pertussis, 32 για B. parapertussis, ενώ κανένα δεν ήταν θετικό για B. holmesii. Το 88% των θετικών περιπτώσεων για B. pertussis ήταν νεογνά μικρότερα του ενός έτους και μόνο 12% ήταν μεταξύ 1 - 14 ετών. Η πλειονότητα των παιδιών ελληνικής εθνικότητας ήταν μικρότερα των 2 μηνών, μην έχοντας φτάσει την ηλικία εμβολιασμού, όπως ορίζεται από το Εθνικό Σύστημα Δημόσιας Υγείας της χώρας μας. Οι μειονότητες των Roma και των μεταναστών αντιπροσωπεύουν ένα μικρό ποσοστό του πληθυσμού της χώρας μας αλλά αποτέλεσαν το 50% του εξετασθέντος πληθυσμού της μελέτης μας. Και οι δύο ομάδες χαρακτηρίζονταν από χαμηλό δείκτη εμβολιαστικής κάλυψης. Ένα ολοκληρωμένο MLVA προφίλ έδωσαν 66 από τα 153 δείγματα που αναλύθηκαν. Ο B. pertussis MLVA type 27 (MT27) (n = 55/66) ήταν ο κυρίαρχος γενετικός τύπος. Το αλλήλιο ptP3 βρέθηκε σε όλα τα δείγματα που εξετάστηκαν με ptP sequencing (n = 25). Συμπεράσματα: H B. pertussis προσβάλλει με μεγάλη συχνότητα ηλικιακές και πληθυσμιακές ομάδες που είναι εμβολιαστικά ακάλυπτες. Oι ομάδες αυτές ήταν ανεμβολίαστα νεογνά < 2 μηνών καθώς και παιδιά Roma και μεταναστών, πιθανώς λόγω έλλειψης εμβολιασμού και χαμηλών κοινωνικοοικονομικών συνθηκών διαβίωσης. Κατά την περίοδο 2010 - 2015, δεν ανιχνεύθηκε καμία περίπτωση B. holmesii. Ο B. pertussis MT27 σε συνδυασμό με τον ptP3 γονότυπο είναι ο κυρίαρχος κλώνος που απαντάται μεταξύ νοσηλευόμενων Ελλήνων, Roma και μεταναστών. Τα αποτελέσματα αυτά είναι παρόμοια με άλλων χωρών που κάνουν χρήση του ακυτταρικού εμβολίου. Η γενετική ποικιλομορφία του μικροβίου είναι μικρή, λόγω της επιλεκτικής πίεσης που ασκούν σε αυτό τα ακυτταρικά εμβόλια, σε συνδυασμό με την υψηλή εμβολιαστική κάλυψη.