Nontypeable Isolates Research Articles

SCC mec typing among the clinical isolates of Methicillin Resistant Staphylococcus aureus (MRSA) from a Tertiary Care Hospital, Puducherry

Introduction: Methicillin resistance is exhibiteddue to presence of mecA gene and is commonly referred as SCCmec types in MRSA. SCCmec typing helps and acts a way to characterize MRSA infections and gain knowledge in its epidemiology. Our study aims to characterize mecA gene responsible in methicillin resistance by SCCmec typing and emphasize the use of SCCmec typing in routine diagnosis for the classification of MRSA. Methods: A total of 191 clinical isolates were included for the study.Documentation like clinical details and associated risk factors for MRSA acquisition were done. SCCmec typing (type I-V) was done by using M-PCR as per Boyeet al., 2007 protocol. Results: In our study, five different SCCmec types namely SCCmec type I, type II, type III, type IV and type V were detected among the clinical isolates of MRSA. Sccmec type V (53%) was found to be the most predominant type followed by SCCmec type III (47%) and SCCmec type IV (37.6%). Among the 191 MRSA isolates, 7 clinical isolates were SCCmec non-typeable isolates. Conclusion: Our study reports existence of multiple SCCmec types among the MRSA clinical isolates. SCCmec typing is helpful in determining the contribution of the isolates in understanding their association being CA-MRSA or HA-MRSA which in turn helps in designing effective surveillance and control strategies in the management of MRSA infections.

Multiple interspecies recombination events documented by whole-genome sequencing in multidrug-resistant Haemophilus influenzae clinical isolates.

Haemophilus influenzae (Hi) was long known as an easy-to-treat bacterium, but increasing resistance against beta-lactams and other critically important antibiotics is now a growing concern. We describe here the whole-genome sequencing (WGS) analysis of three non-typeable Hi isolates received in 2018-2019 by the Belgian National Reference Centre (NRC) for Haemophilus influenzae, as they presented an unusual multi-resistant profile. All three isolates were sequenced by WGS and mapped to the reference isolate Hi Rd KW20. Shorten uptake signal sequences (USSs) known to be associated with homologous recombination were sought in ftsI, murE and murF genes, and inner partial sequences were compared against the blast nucleotide database to look for similarity with other Haemophilus species. Their antimicrobial resistance (AMR) genotype was studied. Core-genome multilocus sequence typing (MLST) was performed on the NTHi database pubMLST to place our isolates in the actual worldwide epidemiology. The isolates also harboured interspecies recombination patterns in the murF-murE-ftsI region involved in cell wall synthesis. The three isolates were multidrug resistant and two of them were also resistant to amoxicillin-clavulanic acid and showed a reduced susceptibility to meropenem. All three isolates belonged to the MLST clonal complex (CC) 422, and WGS revealed that the three were very similar. They harboured mobile genetic elements (carrying blaTEM-1B, mefA and msrD genes associated with resistance), mutations in gyrA and parC linked to fluoroquinolone resistance as well as remodelling events in ompP2 that might be related to lower carbapenem susceptibility. The Hi evolution towards antimicrobial multiresistance (AMR) is a complex and poorly understood phenomenon, although probably linked to a large degree to the presence of USSs and exchange within the family Pasteurellaceae. To better understand the respective roles of clonal expansion, horizontal gene transfers, spontaneous mutations and interspecies genetic rearrangements in shaping Hi AMR, both analysis of Hi communities over time within individuals and worldwide monitoring of non-typeable Hi causing infections should be conducted.

Biofilm Formation by Nontypeable Haemophilus Influenzae and Resistance to Complement-Mediated Clearance.

Biofilm formation has been suggested to be associated with phenotype changes compared with the planktonic form. We screened 1092 Haemophilus influenzae isolates for their genetic relationships and then selected 29 isolates from different genotypes and phenotypes and tested their ability to form biofilm. Our data showed a higher capacity of nontypable isolates, particularly isolates from respiratory and genital infections to form biofilm, compared with typable isolates. This ability to form biofilm was also correlated with reduced deposition of the complement component C3b on biofilm-involved bacteria. These data suggest that the biofilm formation contributes to the virulence of nontypable H. influenzae.

Molecular identification and characterisation of Mannheimia haemolytica

Mannheimia haemolytica is known as one of the major bacterial contributors to Bovine Respiratory Disease (BRD) syndrome. This study sought to establish a novel species-specific PCR to aid in identification of this key pathogen. As well, an existing multiplex PCR was used to determine the prevalence of serovars 1, 2 or 6 in Australia. Most of the 65 studied isolates originated from cattle with a total of 11 isolates from small ruminants. All problematic field isolates in the identification or serotyping PCRs were subjected to whole genome sequencing and bioinformatic analysis. The field isolates were also subjected to rep-PCR fingerprinting. A total of 59 out of the 65 tested isolates were conformed as M. haemolytica by the new species-specific PCR which is based on the rpoB gene. The confirmed M. haemolytica field isolates were assigned to serovars 1 (24 isolates), 2 (seven isolates) and 6 (26 isolates) while two of the isolates were negative in the serotyping PCR. The two non-typeable isolates were assigned to serovar 7 and 14 following whole genome sequencing and bioinformatic analysis. The rep-PCR typing resulted in five major clusters with serovars 1 and 6 often within the same cluster. The M. haemolytica-specific PCR developed in this work was species specific and should be a valuable support for frontline diagnostic laboratories. The serotyping results support the relative importance of serovars 1 and 6 in bovine respiratory disease.

Serotypes, virulence factors and multilocus sequence typing of Glaesserella parasuis from diseased pigs in Taiwan.

Glaesserella parasuis (G. parasuis) belongs to the normal microbiota of the upper respiratory tract in the swine, but virulent strains can cause systemic infections commonly known as Glässer's disease that leads to significant economic loss in the swine industry. Fifteen serotypes of G. parasuis have been classified by gel immunodiffusion test while the molecular serotyping based on variation within the capsule loci have further improved the serotype determination of unidentified field strains. Serovar has been commonly used as an indicator of virulence; however, virulence can be significantly differ in the field isolates with the same serotype. To date, investigations of G. parasuis isolated in Taiwan regarding antimicrobial resistance, serotypes, genotypes and virulence factors remain unclear. A total of 276 G.parasuis field isolates were collected from 263 diseased pigs at the Animal Disease Diagnostic Center of National Chiayi University in Taiwan from January 2013 to July 2021. Putative virulence factors and serotypes of the isolates were identified by polymerase chain reaction (PCR) and antimicrobial susceptibility testing was performed by microbroth dilution assay. Additionally, the epidemiology of G. parasuis was characterized by multilocus sequence typing (MLST). Serotype 4 (33.3%) and 5 (21.4%) were the most prevalent, followed by nontypable isolates (15.9%), serotype 13 (9.4%), 12 (6.5%), 14 (6.2%), 7 (3.3%), 1 (1.8%), 9 (1.1%), 11 (0.7%) and 6 (0.4%). Nine out of 10 putative virulence factors showed high positive rates, including group 1 vtaA (100%), fhuA (80.4%), hhdA (98.6%), hhdB (96.0%), sclB7 (99.6%), sclB11 (94.9%), nhaC (98.2%), HAPS_0254 (85.9%), and cirA (99.3%). According to the results of antimicrobial susceptibility testing, ceftiofur and florfenicol were highly susceptible (>90%). Notably, 68.8% isolates showed multidrug resistance. MLST revealed 16 new alleles and 67 new sequence types (STs). STs of these isolated G. parasuis strains were classified into three clonal complexes and 45 singletons by Based Upon Related Sequence Types (BURST) analysis. All the G. parasuis strains in PubMLST database, including strains from the diseased pigs in the study, were defined into two main clusters by Unweighted Pair Group Method with Arithmetic Mean (UPGMA). Most isolates in this study and virulent isolates from the database were mainly located in cluster 2, while cluster 1 included a high percentage of nasal isolates from asymptomatic carriers. In conclusion, this study provides current prevalence and antimicrobial susceptibility of G. parasuis in Taiwan, which can be used in clinical diagnosis and treatment of Glässer's disease.

A 20-Year Study of Capsular Polysaccharide Seroepidemiology, Susceptibility Profiles, and Virulence Determinants of Klebsiella pneumoniae from Bacteremia Patients in Taiwan.

In this study, we selected bacteremic Klebsiella pneumoniae isolates from the Taiwan Surveillance of Antimicrobial Resistance program. A total of 521 isolates were collected over a period of 2 decades, including 121 from 1998, 197 from 2008, and 203 from 2018. Seroepidemiology showed that the top five capsular polysaccharide types were serotypes K1, K2, K20, K54, and K62, constituting 48.5% of the total isolates, and the respective ratios at each time point have remained similar over the past 2 decades. The antibacterial susceptibility tests showed that K1, K2, K20, and K54 were susceptible to most antibiotics, while K62 was relatively resistant compared to other typeable and nontypeable strains. In addition, six virulence-associated genes, clbA, entB, iroN, rmpA, iutA, and iucA, were predominant in K1 and K2 isolates of K. pneumoniae. In conclusion, serotypes K1, K2, K20, K54, and K62 of K. pneumoniae are the most prevalent serotypes and carry more virulence determinants in bacteremia patients, which may indicate their invasiveness. If further serotype-specific vaccine development is performed, these five serotypes should be included. Since the antibiotic susceptibility profiles were stable over a long duration, empirical treatment may be predicted according to serotype if rapid diagnosis from direct clinical specimens is available, such as PCR or antigen serotyping for serotype K1 and K2. IMPORTANCE This is the first nationwide study to examine the seroepidemiology of Klebsiella pneumoniae using blood culture isolates collected over a period of 20 years. The study found that the prevalence of serotypes remained consistent over the 20-year period, with high-prevalence serotypes associated with invasive types. Nontypeable isolates had fewer virulence determinants than other serotypes. With the exception of serotype K62, the other high-prevalence serotypes were highly susceptible to antibiotics. If rapid diagnosis using direct clinical specimens, such as PCR or antigen serotyping, is available, empirical treatment can be predicted based on serotype, particularly for K1 and K2. The results of this seroepidemiology study could also help the development of future capsule polysaccharide vaccines.

Prevalence and Homology of the Pneumococcal Serine-Rich Repeat Protein at the Global Scale.

Pneumococcal pneumonia remains a WHO high-priority disease despite multivalent conjugate vaccines administered in clinical practice worldwide. A protein-based, serotype-independent vaccine has long-promised comprehensive coverage of most clinical isolates of the pneumococcus. Along with numerous pneumococcal surface protein immunogens, the pneumococcal serine-rich repeat protein (PsrP) has been investigated as a potential vaccine target due to its surface exposure and functions toward bacterial virulence and lung infection. Three critical criteria for its vaccine potential - the clinical prevalence, serotype distribution, and sequence homology of PsrP - have yet to be well characterized. Here, we used genomes of 13,454 clinically isolated pneumococci from the Global Pneumococcal Sequencing project to investigate PsrP presence among isolates, distribution among serotypes, and interrogate its homology as a protein across species. These isolates represent all age groups, countries worldwide, and types of pneumococcal infection. We found PsrP present in at least 50% of all isolates across all determined serotypes and nontypeable (NT) clinical isolates. Using a combination of peptide matching and HMM profiles built on full-length and individual PsrP domains, we identified novel variants that expand PsrP diversity and prevalence. We also observed sequence variability in its basic region (BR) between isolates and serotypes. PsrP has a strong vaccine potential due to its breadth of coverage, especially in nonvaccine serotypes (NVTs) when exploiting its regions of conservation in vaccine design. IMPORTANCE An updated outlook on PsrP prevalence and serotype distribution sheds new light on the comprehensiveness of a PsrP-based protein vaccine. The protein is present in all vaccine serotypes and highly present in the next wave of potentially disease-causing serotypes not included in the current multivalent conjugate vaccines. Furthermore, PsrP is strongly correlated with clinical isolates harboring pneumococcal disease as opposed to pneumococcal carriage. PsrP is also highly present in strains and serotypes from Africa, where the need for a protein-based vaccine is the greatest, giving new reasoning to pursue PsrP as a protein vaccine.

Carriage prevalence, serotype distribution, antimicrobial resistance, pspA typing and pilus islets of Streptococcus pneumoniae isolated from adults living in a Brazilian urban slum.

For Brazilian adults, pneumococcal vaccines have been usually taken only by those who are at higher risk for development of pneumococcal diseases. Since populations from lower socioeconomic status are at high risk of acquiring pneumococcal infections, we investigated the carriage prevalence, colonization risk factors, capsular and surface protein types, and antimicrobial resistance among pneumococcal isolates recovered from adults living in a Brazilian urban slum. Between September-December 2016, we conducted a cross-sectional study among individuals aged≥18years who attended a public primary clinic in Niterói/RJ, Brazil. Pneumococci were isolated by culture on sheep blood agar plates with and without gentamicin. Antimicrobial susceptibility was determined for all isolates. We used PCR to determine capsular types, PspA families (Fam) and pilus islets (PI). Of 385 adults, 32 (8.3%) were pneumococcal carriers. Three carriers had two different pneumococci, totaling 35 isolates. After multivariate analysis, smoking, previous hospitalization, alcohol consumption and co-habitation with children aged<6years increased the odds of pneumococcal carriage, but antibiotic use in the previous 2weeks was found to be a protective factor. Fourteen different serogroups/serotypes were detected and the prevalent ones were 9N/L, 10A, 15B/C and 35F/47F (n=3; 8.6% each). Non-typeable (NT) isolates made up 31.4%. All isolates were susceptible to chloramphenicol, levofloxacin and vancomycin. We found eight (22.9%) penicillin non-susceptible pneumococci (PNSP) with minimum inhibitory concentrations (MICs) of 0.38-1.5μg/mL. The two (5.7%) erythromycin-resistant isolates had MIC>256μg/mL, cMLSB phenotype and the erm(B) gene. Twelve (34.3%) and 17 (48.6%) isolates had PspA Fam1 and Fam2, respectively. Three (8.6%) isolates had genes for pilitwo PI-1 and one PI-2. We detected a low frequency of pneumococcal carriage among the adult population, but a high diversity of serotypes. Frequencies of PNSP and NT isolates resistant to antimicrobial agents are concerning.

Widespread of the Vienna/Hungarian/Brazilian CC8-ST239-SCCmec III MRSA clone in patients hospitalized in the Tunisian Burn and Traumatology Center.

The emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA) in hospitals is a major global public health concern. The current study sought to characterize 25 MRSA clinical isolates collected in a Tunisian hospital from December 2015 to September 2016, with the genetic lineages, virulence factors, and antibiotic resistance mechanisms determined for these isolates. Three spa-types were detected: t037 (23 isolates), t932, and t2235 (one isolate each). Isolates were ascribed to agr I (n=20), agr II (n=1), with four nontypeable isolates. Depending on sequence type (ST), the 25 MRSA isolates were assigned to two clonal complexes (CC8 and CC5), with a predominance of the lineage ST239-CC8 (n=24; 96%). All isolates belonging to CC8 had the SCCmec type III, while the unique CC5 isolate had SCCmec type IV. Antimicrobial susceptibility testing revealed high levels of resistance to aminoglycosides, tetracycline, ciprofloxacin and rifampicin for the majority of isolates belonging to the ST239-CC8 lineage. The ST149-CC5 isolate was susceptible to non-β-lactam antibiotics. One isolate harbored the tsst-1 gene (4%); however, lukS/LukF-PV, eta and etb genes were not detected. The MDR ST239-CC8 clone would seem to be widespread in this hospital. Therefore, a rigorous hygienic control system is urgently required.

Pneumococcal carriage in adults aged 50 years and older in outpatient health care facility during pandemic COVID-19 in Novi Sad, Serbia.

Data related to carriage of Streptococcus pneumoniae (Spn) and antimicrobial resistance patterns in middle-aged and older adults are limited. We assessed the carriage of Spn, and its antibiotic resistance patterns, among participants ≥50 years of age living in the city of Novi Sad during the second year of COVID-19 pandemic. Analysis of prospectively collected data among participants with or without symptoms of upper respiratory tract infection who visited their elected physicians in the Primary Health Care Centre of Novi Sad (outpatient facility) was conducted from May 18, 2021 to December 7, 2021. Both nasopharyngeal (NP) and oropharyngeal (OP) samples from each participant were collected. A total of 1042 samples from 521 study subjects (1 NP and 1 OP sample from each person) were collected. Sixteen samples from the same number of persons (3.1%, 95% confidence interval: 1.76%-4.94%) were culture positive for the presence of Spn. Overall, the median age of study participants was 71 years (range, 50-93 years; 90th percentile, 77 years), and most (197/521, 37.8%) of them were 70-79 years of age. A majority of the study subjects were: females (324/521; 62.2%), sampled during May and June 2021 (376/521, 72.2%), those who did not have contact with children aged 0-10 years in the family (403/521; 77.4%), without smokers in the household (443/521; 85.0%), and those who did not receive vaccine against Spn (519/521; 99.6%). Out of 16 Spn positive samples, for six participants, Spn carriage serotypes were obtained and there were four vaccine (6A, 11A, 15B, and 18C) serotypes, and two (6C and 35F) non-vaccine serotypes. Remaining 10 (62.50%) samples were non-typeable isolates of pneumococci. Among four vaccine serotypes, two (6A and 18C) were represented in PCV13, and 18C along with the other two (11A and 15B) in PPSV23 vaccine. The highest level of resistance of Spn isolates was observed for erythromycin, (10 or 62.50%), and tetracycline, (7 or 43.75%), one isolate showed resistance to penicillin, ampicillin, and amoxicillin/amoxicillin-clavulanic acid, while none of them were resistant to ceftriaxone, trimethoprim/sulfamethoxazole and levofloxacin. There were three multi-drug resistant isolates; one was identified as 6C (non-vaccine serotype), and two other were non-typeable isolates of Spn. In this first study conducted in Serbia on Spn carriage in adults ≥50 years of age, we found low prevalence of Spn carriage and identified 6 serotypes of Spn, four of which were represented in vaccines. These results may support future Spn colonization studies among middle-aged and older adults.

Virulence gene profile and antimicrobial resistance of non-typeable Streptococcus suis isolated from clinically healthy and diseased pigs from North East India.

The present study was conducted to investigate the virulence gene profile and antimicrobial resistance of non-typeable Streptococcus suis isolates circulating in pigs of North East India. Fifty-two non-typeable S. suis isolates from clinically healthy and diseased pigs were screened by using PCR for the presence of the muramidase-released protein (mrp), extracellular factor (epf), hemolysin suilysin (sly), arginine deiminase (arcA), and glutamate dehydrogenase (gdh) genes. Five different virulence gene profiles were observed and the most predominant virulence gene profile found in healthy pigs was mrp- + sly- + arcA- + gdh + + epf- whereas the most predominant virulence gene profile recorded in diseased pigs was mrp+ + sly- + arcA+ + gdh+ + epf-. Significantly lower carrier rate of mrp+ + sly- + arcA+ + gdh+ + epf- virulence gene profile was observed among the isolates from healthy pigs compared to those from diseased pigs (P < 0.05). Antimicrobial resistance patterns of the S. suis isolates revealed fourteen resistance groups (R1 to R14) where 88.46% isolates showed multi-drug resistance. The most predominant resistance pattern observed was CD-COT-E-TE. This is perhaps the first study reporting virulence gene profile and antimicrobial resistance of non-typeable S. suis isolates from pigs in North East India. The occurrence of relatively high levels of resistance of S. suis to some antimicrobials (e.g. macrolides, tetracyclines, and sulphonamides) as observed in the present study may represent a human health concern.

Global Distribution of O Serotypes and Antibiotic Resistance in Extraintestinal Pathogenic Escherichia coli Collected From the Blood of Patients With Bacteremia Across Multiple Surveillance Studies.

Extraintestinal pathogenic Escherichia coli (ExPEC) is the leading cause of bacteremia worldwide, with older populations having increased risk of invasive bacterial disease. Increasing resistance to first-line antibiotics and emergence of multidrug-resistant (MDR) strains represent major treatment challenges. ExPEC O serotypes are key targets for potential multivalent conjugate vaccine development. Therefore, we evaluated the O serotype distribution and antibiotic resistance profiles of ExPEC strains causing bloodstream infections across 4 regions. Blood culture isolates from patients aged ≥60 years collected during 5 retrospective E. coli surveillance studies in Europe, North America, Asia-Pacific, and South America (2011-2017) were analyzed. Isolates were O serotyped by agglutination; O genotyping was performed for nontypeable isolates. Antimicrobial susceptibility testing was also conducted. Among 3217 ExPEC blood culture isolates, the most ubiquitous O serotype was O25 (n = 737 [22.9%]), followed by O2, O6, O1, O75, O15, O8, O16, O4, O18, O77 group, O153, O9, O101/O162, O86, and O13 (prevalence of ≥1%). The prevalence of these O serotypes was generally consistent across regions, apart from South America; together, these 16 O serotypes represented 77.6% of all ExPEC bacteremia isolates analyzed. The overall MDR frequency was 10.7%, with limited variation between regions. Within the MDR subset (n = 345), O25 showed a dominant prevalence of 63.2% (n = 218). Predominant O serotypes among ExPEC bacteremia isolates are widespread across different regions. O25 was the most prevalent O serotype overall and particularly dominant among MDR isolates. These findings may inform the design of multivalent conjugate vaccines that can target the predominant O serotypes associated with invasive ExPEC disease in older adults.

Changes in Invasive Neisseria meningitidis and Haemophilus influenzae Infections in France during the COVID-19 Pandemic.

BackgroundSince the appearance of COVID-19 in January 2020, invasive bacterial infections have decreased significantly worldwide. However, alterations in age and sex distributions, clinical forms, phenotypes, and genotypes of isolates have not been analyzed. Our goal is to present and discuss these data considering the current COVID-19 pandemic situation. Methods: The data of the national reference center for meningococci and Haemophilus influenzae in France were mined to examine the above aspects of invasive bacterial infection before (2018–2019) and after (2020–2021) the COVID-19 pandemic. Detailed epidemiological, clinical, and microbiological data were collected, and whole genome sequencing was carried out on meningococcal isolates (n = 1466). Results: In addition to the overall decline in the number of cases, various changes in age, sex, and phenotypes of isolates were also noted. As for N. meningitidis, more cases were observed in adults, as well as more invasive pneumopathies. Furthermore, fewer hyperinvasive meningococcal genotypes have circulated since COVID-19 emerged. The situation has been different for H. influenzae, as the number of invasive cases among adults decreased due to a reduction in non-typeable isolates. In contrast, cases due to serotypeable isolates, particularly serotypes a and b, increased in children <5 years-old. Conclusions: It is possible that measures implemented to stop COVID-19 may have reduced the circulation of N. meningitidis and H. influenzae isolates, but to a variable extent. This may be due to differences in circulation between these two species according to age groups. Vaccination schedules against these two species may have also influenced the evolution of these invasive bacterial infections since the emergence of the COVID-19 pandemic.

In silico capsule locus typing for serovar prediction of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is a causative agent of pleuropneumonia in pigs of all ages. A . pleuropneumoniae is divided into 19 serovars based on capsular polysaccharides (CPSs) and lipopolysaccharides. The serovars of isolates are commonly determined by serological tests and multiplex PCR. This study aimed to develop a genomic approach for in silico A. pleuropneumoniae typing by screening for the presence of the species-specific apxIV gene in whole-genome sequencing (WGS) reads and identifying capsule locus (KL) types in genome assemblies. A database of the A . pleuropneumoniae KL, including CPS synthesis and CPS export genes, was established and optimized for Kaptive. To test the developed genomic approach, WGS reads of 189 A . pleuropneumoniae isolates and those of 66 samples from 14 other bacterial species were analysed. ariba analysis showed that apxIV was detected in all 189 A . pleuropneumoniae samples. These apxIV-positive WGS reads were de novo assembled into genome assemblies and assessed. A total of 105 A . pleuropneumoniae genome assemblies that passed the quality assessment were analysed by Kaptive analysis against the A . pleuropneumoniae KL database. The results showed that 97 assemblies were classified and predicted as 13 serovars, which matched the serovar information obtained from the literature. The six genome assemblies from previously nontypable isolates were typed and predicted as serovars 17 and 18. Notably, one of the two “Actinobacillus porcitonsillarum” samples was apxIV positive, and its genome assembly was typed as KL03 with high identity and predicted as A . pleuropneumoniae serovar 3. Collectively, a genomic approach was established and could accurately determine the KL type of A . pleuropneumoniae isolates using WGS reads. This approach can be used with high-quality genome assemblies for predicting A . pleuropneumoniae serovars and for retrospective analysis.

Detection and characterization of Escherichia coli associated Oreochromis niloticus sold in the retail markets at Sohag Governorate, Egypt

Fish is a major vehicle for transmission several bacterial diseases to human. Escherichia coli (E. coli) is one of the main causes of food poisoning outbreaks occurring in Egypt due to consumption of contaminated fish and fish products. This study was performed to investigate prevalence of contamination of Oreochromis niloticus (O. niloticus) sold in the retail markets at Sohag Governorate, Egypt with E. coli and Shiga toxin-producing E. coli (STEC) and to determine the antimicrobial susceptibility of the isolates. Therefore, 110 apparently healthy O. niloticus samples were randomly collected from the fish retail markets at Sohag Governorate cities, Egypt and they were bacteriologically examined for presence of E. coli on the surface and in the gills and muscles. Susceptibility of E. coli isolates were tested to 10 different antibiotics and subsequently representative E. coli isolates (n=12) were serotyped and screened by PCR for presence of stx1 and stx2 genes. 22 E. coli isolates were isolated and identified from surface, gills and muscles of the examined O. niloticus with percentage of (8.2%), (10.0%) and (1.8%) respectively, these isolates were isolated from 12 fish only of the examined O. niloticus with total prevalence of 10.9% (12/110). Serotyping revealed that the investigated E. coli isolates belonging to 5 different O-serogroups comprising O55 (33.4%), O125 (25.0%), O26 (8.3%), O76 (8.3%) and O128 (8.3%) in addition to 2 nontypeable isolates (16.7%).

The 13-valent pneumococcal conjugate vaccine (PCV13) does not appear to provide much protection on combined invasive disease due to the six PCV13 non-PCV7 serotypes 1, 3, 5, 6A, 7F, and 19A in Kuwait during 2010–2019

ABSTRACT Kuwait started immunizing children <2 y age with the 7-valent pneumococcal conjugate vaccine, PCV7 from August 2007. PCV7 was replaced by the 13-valent conjugate vaccine, PCV13 from August 2010. In a previous analysis of the results for the period, August 2010–July 2013 (period II), there was no evidence of serotype-specific protection for invasive disease against the additional six serotypes to PCV7 present in PCV13 (non-PCV7 serotypes) as evidenced by isolation from blood and cerebrospinal fluid in any of the age groups, <2 y, 2–5 y, 6–50 y, 51–65 y, and >65 y and all ages, compared to the pre-vaccination period, August 2003–July 2006 (period I). In the current study, we allowed additional time, August 2013–July 2019 (period III) for better vaccine effect and repeated the analysis. We did not find any significant decrease of invasive disease due to the non-PCV7 serotypes of PCV13 in period III and combined II and III periods compared to period I. However, these comparisons showed significant reductions for four of the six and total serotypes of PCV7, and total serotypes of PCV13. Reduction for total PCV13 serotypes was contributed by serotypes of PCV7. It appears that the six non-PCV7 serotypes in PCV13 do not offer much protection. Some contributory factors for the poor effect of the non-PCV7 serotypes may be related to few cases with underpowered statistical analysis, lack of vaccine coverage data, method of vaccine efficacy analysis based on vaccine serotypes relative to all serotypes and unusual rise in non-typeable isolates post vaccination that would have masked true serotypes.

Contamination of chicken carcasses and the abattoir environment with Listeria monocytogenes in Taiwan

ABSTRACT 1. The following study provides the first data on the detection and types of Listeria monocytogenes isolated from broiler chickens during processing and from six Taiwanese abattoir environments. 2. Listeria monocytogenes was not detected in any cloacal (n = 120) or environmental (n = 256) samples collected before and during processing, indicating that faecal material and the environment of abattoirs were not important sources of L. monocytogenes for poultry carcases. However, 28 of 246 (11.4%; 95% CI: 7.7–16.0) rinse samples collected from carcases post-evisceration from three abattoirs were positive for L. monocytogenes. 3. The only serotypes detected were 1/2a (82.1%; 95% CI: 63.1–93.9) and 1/2b (14.3%; 95% CI: 4.0–32.7), with 3.6% (95% CI: 0.1–18.3) non-typable isolates. 4. Characterisation by Pulsed Field Gel Electrophoresis (PFGE) identified five PFGE types, confirming cross-contamination with L. monocytogenes during evisceration, chilling and post-chilling. 5. These findings highlight the potential for cross-contamination to occur through direct contact between carcases, especially whilst in chilling tanks.

Molecular Identification of Invasive Non-typeable Group B Streptococcus Isolates From Denmark (2015 to 2017).

The number of invasive Streptococcus agalactiae (GBS) non-typeable (NT) isolates in Denmark received since 1999 has in general accounted for 10% of all invasive GBS isolates. We present data on 55 clinical NT isolates based on clinical manifestations, clonal relationship, antimicrobial resistance (AMR) determinants, and virulence factors. The GBS isolates included in this study were phenotypic-based NT obtained from 2015 to 2017, as well as 10 reference isolates. Whole genome sequencing (WGS) was performed on all isolates and the data were analyzed for the presence of both species specific genes, capsular genes (genotype), and other relevant genes. We furthermore compared different procedures for detection of serotype specific capsular genes. Overall we were able to genotype 54 of the 55 isolates. After retesting the isolates a phenotype was detected for 20 (36%) isolates, of which the initial phenotyping problem for 13 isolates was found to be due to a problem with serotype Ia specific antiserum. Thirty-five isolates remained phenotypic non-typeable with a majority of genotype V isolates which do not express a capsular gene. From all the Danish invasive GBS isolates from 2015 to 2017, the 35 NT isolates were all detected in the age group above 21 years with bacteremia. The 35 NT isolates belonged to six different well-known human pathogenic clonal complexes. The CDC recommended sequences for capsule genotyping were the most optimal for serotype prediction, because of the sequence simplicity and clear cutoff values. However we recommend to also use other capsular sequences for the NT isolates, if they cannot be genotyped by the CDC method.

Proposal of Actinobacillus pleuropneumoniae serovar 19, and reformulation of previous multiplex PCRs for capsule-specific typing of all known serovars

Two serologically and molecularly non-typeable isolates of the porcine lung pathogen Actinobacillus pleuropneumoniae have been identified from diseased swine in two different continents. Genome sequencing was carried out to identify their diagnostically relevant genotypes. Both isolates are biovar 1 and encode genes for production of ApxIV and ApxII (apxIICA structural genes, and apxIBD export genes). They both possess the same novel type II capsule locus (most similar to serovar 1, but with two capsule genes not previously found in A. pleuropneumoniae) but differ in their O-Ag loci. Strain 7213384-1 from Denmark, which we propose as the reference strain for serovar 19, has a serogroup 3/6/8/15 O-Ag locus; the Canadian isolate A08-013 has a serogroup 4/7 O-Ag locus. We have expanded the second of our two previously described A. pleuropneumoniae mPCRs to include capsule gene-specific primers for definitive detection of serovars 13–14 and 16–19.

Ct value-based real time PCR serotyping of Glaesserella parasuis

Glaesserella parasuis is the causative agent of Glässer's disease in swine. Serotyping plays an essential role in prevalence investigations and in the development of vaccination strategies for the prevention of this disease. Molecular serotyping based on variation within the capsule loci of the 15 serovars is more accurate and efficient than traditional serological serotyping. To reduce the running time and facilitate ease of data interpretation, we developed a simple and rapid cycle threshold (Ct) value-based real time PCR (qPCR) method for the identification and serotyping of G. parasuis. The qPCR method distinguished between all 15 serovar reference strains of G. parasuis with efficiency values ranging between 85.5 % and 110.4 % and, R2 values > 0.98. The qPCR serotyping was evaluated using 83 clinical isolates with 43 of the isolates having been previously assigned to a serovar by the gel immuno-diffusion (GID) assay and 40 non-typeable isolates. The qPCR results of 41/43 (95.3 %) isolates were concordant with the GID assay except two isolates of serovar 12 were assigned to serovar 5. In addition, the qPCR serotyping assigned a serovar to each of the 40 non-typeable isolates. Of the 83 isolates tested to assign a serovar, a concordance rate of 98.8 % (82/83) was determined between the qPCR and the previously reported multiplex PCR of Howell et al. (2015) (including those that were either serovars 5 or 12). Despite the inability to differentiate between serovars 5 and 12, the Ct value-based qPCR serotyping represents an attractive alternative to current molecular serotyping method for G. parasuis and could be used for both epidemiological monitoring and the guidance of vaccination programs.