Infant Pneumococcal Carriage in Belgium Not Affected by COVID-19 Containment Measures

Streptococcus pneumoniae is an important and frequently carried respiratory pathogen that has the potential to cause serious invasive diseases, such as pneumonia, meningitis, and sepsis. Young children and older adults are among the most vulnerable to developing serious disease. With the arrival of the COVID-19 pandemic and the concomitant restrictive measures, invasive disease cases caused by respiratory bacterial species, including pneumococci, decreased substantially. Notably, the stringency of the containment measures as well as the visible reduction in the movement of people appeared to coincide with the drop in invasive disease cases. One could argue that wearing protective masks and adhering to social distancing guidelines to halt the spread of the SARS-CoV-2 virus, also led to a reduction in the person-to-person transmission of respiratory bacterial species. Although plausible, this conjecture is challenged by novel data obtained from our nasopharyngeal carriage study which is performed yearly in healthy daycare center attending children. A sustained and high pneumococcal carriage rate was observed amid periods of stringent restrictive measures. This finding prompts us to revisit the connection between nasopharyngeal colonization and invasion and invites us to look closer at the nasopharyngeal microbiome as a whole.

High Carriage Rate of the Multiple Resistant Plasmids Harboring Quinolone Resistance Genes in Enterobacter spp. Isolated from Healthy Individuals

Antimicrobial-resistant bacteria causing intractable and even fatal infections are a major health concern. Resistant bacteria residing in the intestinal tract of healthy individuals present a silent threat because of frequent transmission via conjugation and transposition. Plasmids harboring quinolone resistance genes are increasingly detected in clinical isolates worldwide. Here, we investigated the molecular epidemiology of plasmid-mediated quinolone resistance (PMQR) in Gram-negative bacteria from healthy service trade workers. From 157 rectal swab samples, 125 ciprofloxacin-resistant strains, including 112 Escherichia coli, 10 Klebsiella pneumoniae, two Proteus mirabilis, and one Citrobacter braakii, were isolated. Multiplex PCR screening identified 39 strains harboring the PMQR genes (including 17 qnr,19 aac(6′)-Ib-cr, and 22 oqxA/oqxB). The genome and plasmid sequences of 39 and 31 strains, respectively, were obtained by short- and long-read sequencing. PMQR genes mainly resided in the IncFIB, IncFII, and IncR plasmids, and coexisted with 3–11 other resistance genes. The high PMQR gene carriage rate among Gram-negative bacteria isolated from healthy individuals suggests the high-frequency transmission of these genes via plasmids, along with other resistance genes. Thus, healthy individuals may spread antibiotic-resistant bacterial, highlighting the need for improved monitoring and control of the spread of antibiotic-resistant bacteria and genes in healthy individuals.

Investigation of extramammary sources of Group B Streptococcus reveals its unusual ecology and epidemiology in camels

Camels are vital to food production in the drylands of the Horn of Africa, with milk as their main contribution to food security. A major constraint to camel milk production is mastitis, inflammation of the mammary gland. The condition negatively impacts milk yield and quality as well as household income. A leading cause of mastitis in dairy camels is Streptococcus agalactiae, or group B Streptococcus (GBS), which is also a commensal and pathogen of humans and cattle. It has been suggested that extramammary reservoirs for this pathogen may contribute to the occurrence of mastitis in camels. We explored the molecular epidemiology of GBS in camels using a cross-sectional study design for sample collection and phenotypic, genomic and phylogenetic analysis of isolates. Among 88 adult camels and 93 calves from six herds in Laikipia County, Kenya, GBS was detected in 20% of 50 milk samples, 25% of 152 nasal swabs, 8% of 90 oral swabs and 3% of 90 rectal swabs, but not in vaginal swabs. Per camel herd, two to four sequence types (ST) were identified using Multi Locus Sequence Typing (MLST). More than half of the isolates belonged to ST617 or its single-locus variant, ST1652, with these STs found across all sample types. Capsular serotype VI was detected in 30 of 58 isolates. In three herds, identical STs were detected in milk and swab samples, suggesting that extramammary sources of GBS may contribute to the maintenance and spread of GBS within camel herds. This needs to be considered when developing prevention and control strategies for GBS mastitis. The high nasal carriage rate, low recto-vaginal carriage rate, and high prevalence of serotype VI for GBS in camels are in stark contrast to the distribution of GBS in humans and in cattle and reveal hitherto unknown ecological and molecular features of this bacterial species.

Molecular surveillance of pneumococcal carriage following completion of immunization with the 13-valent pneumococcal conjugate vaccine administered in a 3 + 1 schedule

In a cross-sectional study, with the use of molecular methods, we aimed to gain insight into oropharyngeal pneumococcal colonization over time in 1212 Greek children recruited in general pediatric settings throughout the country; they were fully vaccinated with PCV13 (3 + 1 schedule). A single sample was obtained from each child at a time interval of 26 days to 70 months after administration of the 4th (booster) PCV13 dose; sampling time was divided into six time intervals. Carriage of Streptococcus pneumoniae was detected by real-time PCR targeting the lytA gene and isolates were serotyped by singleplex real-time PCR assays. Multiple control procedures to avoid false-positive results were applied. We showed an overall S. pneumoniae carriage rate of 48.6%. Serotyping identified typeable isolates in 82% of the total lytA-positive samples. Non-PCV13 serotypes represented 83.8% of total isolates when excluding serogroups with mixed PCV13 and non-PCV13 serotypes. In multivariate analysis daycare/school attendance emerged as the main contributing factor. Notably, serotypes 19A and 3 were the only two PCV13 serotypes the colonization rate of which increased over time (χ2 for trend P < 0.001 and P = 0.012, respectively). The application of the SP2020 gene on lytA-positive serotyped samples showed pneumococcal colonization in 97% of cases, and the overall colonization profile over time closely resembled that of the lytA gene. With the provisions of the methodological approach and age group of our study, the use of the oropharynx emerges as a reliable alternative to the nasopharynx in estimating pneumococcal carriage in epidemiological studies.

Clostridioides Difficile Carriage Rate Increases during Pediatric ALL Treatment

Background Clostridioides difficile infection (CDI) is frequent in pediatric patients with acute lymphoblastic leukemia (ALL). Studies have shown upwards of 20% positivity rate in CDI testing among pediatric oncology patients, and up to several percent in pediatric ALL patients in the first 180 days of diagnosis. Antibiotic usage has been variably linked to CDI positivity in these populations. As CDI testing is usually done in symptomatic patients, the question of C. difficile carriage versus CDI has not been addressed. We and others have shown that microbiome is altered in pediatric ALL patients and survivors. We conducted a longitudinal stool microbiome study in pediatric ALL patients and tested the hypothesis that alteration of the microbiome during ALL treatment promotes C. difficile carriage. Methods Children with ALL were prospectively recruited on a rolling basis and stool samples were collected at diagnosis (Dx) and at the end of induction (EOI), consolidation (EOC), interim maintenance I (IMI), delayed intensification (DI), interim maintenance II (IMII), as well as approximately 3 months and 6 months into maintenance (M3, M6). Stool samples from healthy siblings were used as controls. TaqMan-based quantitative-PCR (qPCR) was performed on DNA extracted from stool samples to detect C. diff 16S rRNA, tcdA, (Toxin A) and tcdB (Toxin B) genes . Samples positive or either tcdA or tcdB, or both, were designated positive for toxigenic C. difficile. 16S rRNA hypervariable region V4 was sequenced and analyzed for microbiome diversity and relative abundance of microbiota. Results 32 ALL patients age 3 months-19 years were included. The diagnoses were 12 standard risk and 14 high risk pre-B ALL, 5 T-ALL, and 1 relapsed pre-B ALL. Stool samples were collected from 18 healthy siblings. The numbers of samples tested at each treatment phase were: 29 Dx, 24 EOI, 23 EOC, 25 IMI, 21 DI, 6 IMII, 14 M3, 7 M6. No patient had symptoms suggestive of CDI, and no patient was clinically tested or treated for CDI. Total number of stool samples tested was 149, of which 43 (29%) were positive for toxigenic C. difficile (Figure 1). At diagnosis, 2/29 (7%) patients were positive, compared to 2/18 (11%) in healthy siblings. At EOI, positivity rate increased to 17%, then up to 40% - 52% at EOC, IMI, and DI. C. difficile positivity were lower around 30% at M3 and M6, although few patients reached maintenance to contribute samples for analysis. Twenty-five patients (78%) were positive at some phase. Longitudinal analysis of individual patients showed that C. difficile positivity was intermittent through treatment phases; only 3 patients remained persistently positive. Seven patients (22%) were never positive. Multivariate analysis showed that EOC, IMI, and DI treatment phases were significant risk factors for C. difficile carriage. Neither the number of antibiotics nor the number of antibiotic courses administered was significant. Leukemia risk stratification (high risk versus standard risk) also did not correlate with C. difficile positivity. Microbiome analysis showed statistically significant differences in relative abundance of certain taxa between C. diff positive and negative samples at the class, order, and family levels (Figure 2). Examples include depletion of the class Verrucomicrobiae, which contains protective Akkermansia, and depletion of the common taxa Bifidobacteriaceae and Ruminococcaceae. Conclusion Longitudinal PCR testing of toxigenic C. difficile in pediatric ALL patients demonstrated increased C. difficile prevalence further into treatment phases. C. difficile carriage correlated significantly with depletion of several bacterial taxa, as microbiome diversity decreased overall with successive treatment phases. Our data lend support to the hypothesis that altered microbiome in ALL treatment allows permissibility for C. difficile carriage. In addition, no C. difficile positive patient had symptoms of CDI, therefore, caution must be taken in clinical testing, as there is a high asymptomatic carriage rate. Further longitudinal testing during maintenance and off-therapy is needed to see if C. difficile carriage rate returns to baseline and correlates with recovery of gut microbiome. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Prevalence of Pneumococcal Carriage among Jordanian Infants in the First 6 Months of Age, 2008–2016

Background: Streptococcus pneumoniae is an opportunistic human-adapted pathogen driven by nasopharyngeal carriage. Aims: To find the pneumococcal carriage rate, resistance, serotypes, and coverage of pneumococcal conjugate vaccines (PCVs) among infants in the first six months of age in the period from March 2008 to April 2016. Methods: Nasopharyngeal swabs (NP) were taken from healthy infants from the northern part of Jordan. Swabs were processed for cultivation, identification, resistance testing and serotyping according to standard methods. Results: During the surveillance period, 484 infants of this age group were tested, with a total carriage rate of 56.2%. 96.2% of infants one to two months of age got one PCV7 injection and were 58% carriers at the time of the first injection. At age three to four months, 84.9% had received two injections, with a carriage rate of 54.9% at the time of the second injection. At ages five to six months, 12.5% had received one to three injections, with a carriage rate of 43.8%. Predominant serotypes in all age groups were 19F (12.5%), 6A (11.4%), 11A (8.4%), 19A (7.0%), 6B (6.6%), 23F (5.9%), 15B (5.1%), 15A and 23A (4.0% each). Coverage of PCV7, PCV13 and the future PCV20 among all cases were 30.5%, 50.7% and 70.6%, respectively. The highest coverage rate of 78.6% was noticed in the age group at five to six months with the future PCV20. Antibiotic resistance was the highest in the first age group. Conclusions: Pneumococcal carriage starts from the first month of the infant’s life. The highest coverage was noticed for PCV20, which implies the necessity for inoculation with future vaccines.

Antibiotic-resistant Gram-negative Bacteria in Urban Flies, and the Increased Risk Posed by Open-air Markets in Mexico City

Aims: Flies are known to spread antibiotic resistant bacteria (ARB), especially from farms to cities; but they may also play a role in the intra-urban dispersion of ARB, in conjunction with poor sanitary conditions. Here, we characterized gram-negative ARB isolated from urban flies (Lucilia and Sarcophaga spp.), and the co-relation with the periodic installation of two open-air markets in Mexico City. Methodology: Forty-two flies were individually captured, and 116 gram-negatives (68 of them Escherichia coli) were isolated from them. Resistance prevalence, and the presence of class 1 integrons was assessed. Results: The isolates were resistant to an average of 2.26 antibiotics (2.6 for E. coli), and 33% of E. coli isolates carried the intI1 gene. Thirteen percent of E. coli isolates produced extended-spectrum beta-lactamases (ESBL), all of them CTX-M, alone or, mostly, along TEM enzymes. Comparing data from market-free days vs. days when open-air markets were installed, the average number of resistance phenotypes per E. coli isolate went from 2.14 to 3.09; the number of resistance phenotypes per fly from 4.62 to 8.88; the average number of resistances per isolate per fly from 1.25 to 2.43; and the ESBL-producing carriage rate per fly from 0.08 to 0.38, respectively (P <.05). Other resistance parameters, were consistently higher among flies captured on market days, but differences were not significant. Conclusion: Urban flies in Mexico City carry a high number of gram-negative ARB; the presence of open-air markets significantly increase the risk of fly-mediated ARB spreading to the neighboring areas.

Nasopharyngeal Meningococcal Carriage among Children and Adolescents in Turkey in 2018: An Unexpected High Serogroup X Carriage

Meningococcal carriage studies and transmission modeling can predict IMD epidemiology and used to define invasive meningococcal disease (IMD) control strategies. In this multicenter study, we aimed to evaluate the prevalence of nasopharyngeal Neisseria meningitidis (Nm) carriage, serogroup distribution, and related risk factors in Turkey. Nasopharyngeal samples were collected from a total of 1267 children and adolescents and were tested with rt-PCR. Nm carriage was detected in 96 participants (7.5%, 95% CI 6.1–9.0), with the peak age at 13 years (12.5%). Regarding age groups, Nm carriage rate was 7% in the 0–5 age group, was 6.9%in the 6–10 age group, was 7.9% in the 11–14 age group, and was 9.3% in the 15–18 age group. There was no statistically significant difference between the groups (p > 0.05). The serogroup distribution was as follows: 25% MenX, 9.4% MenA, 9.4% MenB, 2.1% MenC, 3.1% MenW, 2.1% for MenY, and 48.9% for non-groupable. The Nm carriage rate was higher in children with previous upper respiratory tract infections and with a high number of household members, whereas it was lower in children with antibiotic use in the last month (p < 0.05 for all). In this study, MenX is the predominant carriage strain. The geographical distribution of Nm strains varies, but serogroup distribution in the same country might change in a matter of years. Adequate surveillance and/or a proper carriage study is paramount for accurate/dynamic serogroup distribution and the impact of the proposed vaccination.

Detection of Clostridioides Difficile Toxin B Gene: Benefits of Identifying Gastrointestinal Pathogens by mPCR Assay in the Diagnosis of Diarrhea in Pediatric Patients

In the pediatric population, severe Clostridioides difficile infection sometimes occurs, but most cases are asymptomatic. Since the asymptomatic carriage rate is reportedly high in pediatric populations, diagnosis of CDI is difficult. Here, we analyzed 960 results of gastrointestinal pathogen multiplex PCR to estimate the positive rate of toxigenic C. difficile in pediatric populations aged between 0 and 18 years. The overall rate of C. difficile toxin B positivity was 10.1% in the stool samples. The positive rate peaked in 1-year-old infants (29/153, 19.0%), and decreased continually thereafter. The positive rate we observed was lower than the rates described in the literature. Remarkably, no C. difficile was detected in neonates. Antibiotic usage was inversely related to the positive rate, especially in infants < 2 years of age. The odds ratio of antibiotics was 0.44 (95% confidence interval (CI) 0.28–0.68; P < 0.001). The presence of concomitant gastrointestinal pathogens was not associated with toxigenic C. difficile positivity. Even though toxigenic C. difficile infection is neither an important nor a common cause of pediatric diarrhea, children can spread it to adults who are at risk of developing CDI. Pediatric population can act as hidden reservoirs for pathogenic strains in the community.

Carriage of Streptococcus Pneumoniae In Unvaccinated Toddlers At The Time of Pneumococcal Conjugate Vaccine Implementation Into The National Immunization Program In Poland

We investigated pneumococcal carriage among unvaccinated children under five years of age at the time of conjugate polysaccharide vaccine (PCV) introduction into the national immunization program (NIP). Paired nasopharyngeal swab (NPS) and saliva samples collected between 2016 and 2020 from n=394 children were tested with conventional culture and using qPCR. The carriage rate detected by culture was 25.4% (97 of 394), by qPCR 39.1% (155 of 394), and 40.1% (158 of 394) overall. The risk of carriage was significantly elevated among day care center attendees, and during autumn/winter months. Among strains cultured, the most common serotypes were: 23A, 6B, 15BC, 10A, 11A. The coverage of PCV10 and PCV13 was 23.2% (23 of 99) and 26.3% (26 of 99), respectively. Application of qPCR lead to detection of 168 serotype carriage events, with serogroups 15, 6, 9 and serotype 23A most commonly detected. Although the highest number of carriers was identified by testing NPS with qPCR, saliva significantly contributed to the overall number of detected carriers. Co-carriage of multiple serotypes was detected in 25.3% (40 of 158) of carriers. Results of this study represent a baseline for the future surveillance of effects of pneumococcal vaccines in NIP in Poland.