Comparison of serological and molecular methods for differentiation between genotype A and genotype B strains of small ruminant lentiviruses.

Introduction: Small ruminant lentiviruses (SRLV) cause multisystemic, degenerative and chronic disease in sheep and goats. There are five genotypes (A, B, C, D and E), of which A and B are the most widespread. The purpose of this study was to evaluate the serotyping efficiency of the Eradikit SRLV Genotyping ELISA and the molecular typing efficiency of a newly developed nested real-time PCR targeting the long terminal repeat-gag (LTR-gag) region using samples from animals infected with subtypes of SRLV known to circulate in Poland. Material and Methods: A total of 97 sera samples taken from 34 sheep and 63 goats were immunoassayed, and 86 DNA samples from 31 sheep and 55 goats were tested with the PCR. All ruminants were infected with known SRLV strains of the A1, A5, A12, A13, A16, A17, A18, A23, A24, A27, B1 and B2 subtypes. Results: A total of 69 (80.2%, 95% confidence interval 71.6%-88.8%) out of 86 tested samples gave positive results in the PCR. In 17 out of the 86 (19.8%) samples, no proviral DNA of SRLV was detected. The differentiation between MVV (genotype A) and CAEV (genotype B) by PCR matched the predating phylogenetic analysis invariably. No cross-reactivity was observed. On the other hand, the proportion of samples genotyped the same by the older phylogenetic analysis and the Eradikit SRLV Genotyping ELISA was 42.3%. The test was unable to classify 40.2% of samples, and 17.5% of sera were incorrectly classified. Conclusion: Our results showed that the Eradikit SRLV genotyping kit is not a reliable method for predicting SRLV genotype, while the nested real-time PCR based on the LTR-gag region did prove to be, at least for genotypes A and B.

In Silico Typing and Identification Confirmation of Isolates.

Streptococcus suis is an important zoonotic pathogen causing severe infections in pigs and humans. Serotyping of S. suis strains is crucial for epidemiological surveillance, outbreak investigations, and understanding the pathogenesis of this bacterium. Here, we describe a step-by-step approach that enhances a previously developed pipeline by utilizing a computational script for efficient and accurate typing of S. suis strains. The pipeline is implemented in Perl programming language and leverages the Short Read Sequence Typing for Bacterial Pathogens (SRST2) tool. It integrates various bioinformatics techniques and utilizes multiple databases, including a serotype database, cpsH confirmation database, multi-locus sequence typing (MLST) database, recN species-specific gene database, and virulence gene database. These databases contain comprehensive information on S. suis serotypes, genetic markers, and virulence factors. The script can utilize paired-end or single-end fastq files as input and first confirms the species by sequence read data aligning to the recN gene, ensuring the accurate identification of S. suis strains. The pipeline next performs MLST typing and virulence factor identification using SRST2 while in a parallel processes it performs in silico serotyping of the strains. The pipeline offers a streamlined and semiautomated approach to serotyping S. suis strains, facilitating large-scale studies and reducing the manual effort required for data analysis.

Emergence of Bluetongue Virus Serotype 3, the Netherlands, September 2023.

Since 1998, notifiable bluetongue virus (BTV) serotypes 1-4, 6, 8, 9, 11, and 16 have been reported in Europe. In August 2006, a bluetongue (BT) outbreak caused by BTV serotype 8 began in northwestern Europe. The Netherlands was declared BT-free in February 2012, and annual monitoring continued. On September 3, 2023, typical BT clinical manifestations in sheep were notified to the Netherlands Food and Product Safety Consumer Authority. On September 6, we confirmed BTV infection through laboratory diagnosis; notifications of clinical signs in cattle were also reported. We determined the virus was serotype 3 by whole-genome sequencing. Retrospective analysis did not reveal BTV circulation earlier than September. The virus source and introduction route into the Netherlands remains unknown. Continuous monitoring and molecular diagnostic testing of livestock will be needed to determine virus spread, and new prevention strategies will be required to prevent BTV circulation within the Netherlands and Europe.

Detection of Helicobacter pylori strain types and analysis of risk factors among subjects from Hainan Province, China.

OBJECTIVE: To explore the prevalence of type I and type II Helicobacter pylori infection and investigate risk factors in a population from Hainan Province in China. METHODS: Data came from a large, cross-sectional study conducted from August 2022 to April 2023 involving five cities of Hainan. Subjects with confirmed 14C-urea breath test (UBT) and positive serological assay were included. All subjects had a gastroscopy. According to presence or absence of CagA/VacA proteins, subjects were classified as either type I (present) or type II strains (absent). Gastroscopic findings and several socio-demographic factors were examined for correlation with antibody serotyping. RESULTS: In total, 410 subjects were investigated for H. pylori strain types. The overall prevalence of the highly virulent, type I H. pylori strain was 79% (324/410) and type II strain was 21% (86/410). There was a strong association between type I strain and peptic ulcer disease. Of several sociodemographic factors investigated, only smoking and data over baseline (DOB) values showed significant differences between type 1 and type II strains. Logistic regression analysis showed a lower risk of type I H. pylori infection in smokers compared with non-smokers, and a higher risk of H. pylori type I infection in subjects with medium and high data over baseline (DOB) values compared with subjects who had low DOB values. CONCLUSION: Highly virulent, type I H. pylori infections predominate in Hainan and the co-positivity of CagA and VacA antibodies are related to type I H. pylori infection. We found that Type I H. pylori was closely associated with peptic ulcer disease and the DOB values were generally high.

Designing a Multiplex PCR-xMAP Assay for the Detection and Differentiation of African Horse Sickness Virus, Serotypes 1-9.

African horse sickness is a severe and often fatal disease affecting all species of equids. The aetiological agent, African horse sickness virus (AHSV), can be differentiated into nine serotypes. The identification of AHSV serotypes is vital for disease management, as this can influence vaccine selection and help trace disease incursion routes. In this study, we report the development and optimisation of a novel, molecular-based assay that utilises multiplex PCR and microsphere-based technology to expedite detection and differentiation of multiple AHSV serotypes in one assay. We demonstrated the ability of this assay to identify all nine AHSV serotypes, with detection limits ranging from 1 to 277 genome copies/µL depending on the AHSV serotype. An evaluation of diagnostic sensitivity and specificity revealed a sensitivity of 88% and specificity of 100%. This method can serotype up to 42 samples per run and can be completed in approximately 4-6 h. It provides a powerful tool to enhance the rapidity and efficiency of AHSV serotype detection, thereby facilitating the generation of epidemiological data that can help understand and control the incidence of AHSV worldwide.

Virulence and Antimicrobial Resistance of Listeria monocytogenes Isolated from Ready-to-Eat Food Products in Romania.

Listeria monocytogenes (L. monocytogenes) poses a significant threat to food safety due to its ability to cause severe human illness and its resistance to various antibiotics and environmental conditions. This study investigated the prevalence, serotype distribution, virulence gene profiles, and antimicrobial resistance patterns of L. monocytogenes in ready-to-eat (RTE) food products from Romania. A total of 8151 samples were analyzed, including various processed dairy, bovine, poultry, pork, and fish products. Bacterial isolation was conducted using the classical standard method, followed by confirmation through biochemical and molecular testing. Among the isolated strains, serotypes 1/2a, 1/2b, and 1/2c were identified, with a prevalence of 75% for serotype 1/2a. Additionally, virulence genes specific to listeriolysin O (hlyA) and regulatory factor A (prfA) were detected in all isolates. Antimicrobial susceptibility testing revealed varying resistance patterns among the L. monocytogenes strains. Trimethoprim-sulfamethoxazole and oxacillin showed the highest prevalence of resistance at 26.92% and 23.07%, respectively. However, all strains remained susceptible to ciprofloxacin, levofloxacin, and moxifloxacin. Notably, 23.07% of the isolates exhibited multidrug resistance, with the most common pattern being resistance to oxacillin, penicillin, and tetracycline. Analysis of antimicrobial resistance genes identified tetracycline resistance genes, particularly tet(C), tet(M), and tet(K), in a significant proportion of isolates. The presence of ampC and dfrD genes was also notable, indicating potential mechanisms of resistance. These results emphasize the necessity for ongoing surveillance of L. monocytogenes in RTE foods and emphasize the importance of thorough monitoring of antimicrobial resistance to guide public health strategies within the European Union.

A Proof-of-Concept Protein Microarray-Based Approach for Serotyping of Salmonella enterica Strains.

Salmonella enterica, a bacterium causing foodborne illnesses like salmonellosis, is prevalent in Europe and globally. It is found in food, water, and soil, leading to symptoms like diarrhea and fever. Annually, it results in about 95 million cases worldwide, with increasing antibiotic resistance posing a public health challenge. Therefore, it is necessary to detect and serotype Salmonella for several reasons. The identification of the serovars of Salmonella enterica isolates is crucial to detect and trace outbreaks and to implement effective control measures. Our work presents a protein-based microarray for the rapid and accurate determination of Salmonella serovars. The microarray carries a set of antibodies that can detect different Salmonella O- and H-antigens, allowing for the identification of multiple serovars, including Typhimurium and Enteritidis, in a single miniaturized assay. The system is fast, economical, accurate, and requires only small sample volumes. Also, it is not required to maintain an extensive collection of sera for the serotyping of Salmonella enterica serovars and can be easily expanded and adapted to new serovars and sera. The scientific state of the art in Salmonella serotyping involves the comparison of traditional, molecular, and in silico methods, with a focus on economy, multiplexing, accuracy, rapidity, and adaptability to new serovars and sera. The development of protein-based microarrays, such as the one presented in our work, contributes to the ongoing advancements in this field.

Posibilities for use of whole genome sequencing (WGS) for the analysis of Streptococcus pneumoniae isolates.

Streptococcus pneumoniae (pneumococcus) is a Gram-positive coccus causing both non-invasive and invasive infectious diseases. Pneumococcal diseases are vaccine preventable. Invasive pneumococcal diseases (IPD) meeting the international case definition are reported nationally and internationally and are subject to surveillance programmes in many countries, including the Czech Republic. An important part of IPD surveillance is the monitoring of causative serotypes and their frequency over time and in relation to ongoing vaccination programmes. In the world and in the Czech Republic, whole genome sequencing (WGS) is increasingly used for pneumococci, which allows for serotyping from sequencing data, precise analysis of their genetic relationships, and the study of genes present in their genome. Whole-genome sequencing enables the generation of reliable and internationally comparable data that can be easily shared. Sequencing data are analysed using bioinformatics tools that require knowledge in the field of natural sciences with an emphasis on genetics and expertise in bioinformatics. This publication presents some options for pneumococcal analysis, i.e., serotyping, multilocus sequence typing (MLST), ribosomal MLST (rMLST), core genome MLST (cgMLST), whole genome MLST (wgMLST), single nucleotide polymorphism (SNP) analysis, assignment to Global Pneumococcal Sequence Cluster (GPSC), and identification of virulence genes and antibiotic resistance genes. The WGS strategies and applications for Europe and WGS implementation in practice are presented. WGS analysis of pneumococci allows for improved IPD surveillance, thanks to molecular serotyping, more detailed typing, generation of internationally comparable data, and improved evaluation of the effectiveness of vaccination programmes.

A narrative review of genomic characteristics, serotype, immunogenicity, and vaccine development of Streptococcus pneumoniae capsular polysaccharide.

This narrative review describes genomic characteristic, serotyping, immunogenicity, and vaccine development of Streptococcus pneumoniae capsular polysaccharide (CPS). CPS is a primary virulence factor of S. pneumoniae. The genomic characteristics of S. pneumoniae CPS, including the role of biosynthetic gene and genetic variation within cps (capsule polysaccharide) locus which may lead to serotype replacement are still being investigated. One hundred unique serotypes of S. pneumoniae have been identified through various methods of serotyping using phenotypic and genotypic approach. The advantages and limitations of each method are various, emphasizing the need for accurate and comprehensive serotyping for effective disease surveillance and vaccine targeting. In addition, we elaborate the critical role of CPS in vaccine development by providing an overview of immunogenicity, ongoing research of pneumococcal vaccines, and the impact on disease burden.

A combination of GRA3, GRA6 and GRA7 peptides offer a useful tool for serotyping type II and III Toxoplasma gondii infections in sheep and pigs.

The clinical consequences of toxoplasmosis are greatly dependent on the Toxoplasma gondii strain causing the infection. To better understand its epidemiology and design appropriate control strategies, it is important to determine the strain present in infected animals. Serotyping methods are based on the detection of antibodies that react against segments of antigenic proteins presenting strain-specific polymorphic variations, offering a cost-effective, sensitive, and non-invasive alternative to genotyping techniques. Herein, we evaluated the applicability of a panel of peptides previously characterized in mice and humans to serotype sheep and pigs. To this end, we used 51 serum samples from experimentally infected ewes (32 type II and 19 type III), 20 sheep samples from naturally infected sheep where the causative strain was genotyped (18 type II and 2 type III), and 40 serum samples from experimentally infected pigs (22 type II and 18 type III). Our ELISA test results showed that a combination of GRA peptide homologous pairs can discriminate infections caused by type II and III strains of T. gondii in sheep and pigs. Namely, the GRA3-I/III-43 vs. GRA3-II-43, GRA6-I/III-213 vs. GRA6-II-214 and GRA6-III-44 vs. GRA6-II-44 ratios showed a statistically significant predominance of the respective strain-type peptide in sheep, while in pigs, in addition to these three peptide pairs, GRA7-II-224 vs. GRA7-III-224 also showed promising results. Notably, the GRA6-44 pair, which was previously deemed inefficient in mice and humans, showed a high prediction capacity, especially in sheep. By contrast, GRA5-38 peptides failed to correctly predict the strain type in most sheep and pig samples, underpinning the notion that individual standardization is needed for each animal species. Finally, we recommend analyzing for each animal at least 2 samples taken at different time points to confirm the obtained results.

International circumpolar surveillance: update on the interlaboratory quality control program for Streptococcus pneumoniae, 2009 to 2020.

The International Circumpolar Surveillance (ICS) program is a population-based surveillance network for invasive bacterial diseases throughout Arctic countries and territories. The ICS quality control program for Streptococcus pneumoniae serotyping and antimicrobial susceptibility testing has been ongoing since 1999. Current participating laboratories include the Provincial Laboratory for Public Health in Edmonton, Alberta; Laboratoire de santé publique du Québec in Sainte-Anne-de-Bellevue, Québec; the Centers for Disease Control's Arctic Investigations Program in Anchorage, Alaska; the Neisseria and Streptococcus Reference Laboratory at Statens Serum Institut in Copenhagen, Denmark; the Department of Clinical Microbiology, Landspitali in Reykjavik, Iceland; and Public Health Agency of Canada's National Microbiology Laboratory in Winnipeg, Manitoba. From 2009 to 2020, 140 isolates of S. pneumoniae were distributed among the six laboratories as part of the quality control program. Overall serotype concordance was 96.9%, with 99.3% concordance to pool level. All participating laboratories had individual concordance rates >92% for serotype and >97% for pool. Overall concordance by modal minimum inhibitory concentration (MIC) for testing done by broth microdilution or Etest was 99.1%, and >98% for all antimicrobials tested. Categorical concordance was >98% by both CLSI and EUCAST criteria. For two laboratories performing disc diffusion, rates of concordance by modal MIC were >97% for most antimicrobials, except chloramphenicol (>93%) and trimethoprim/sulfamethoxazole (>88%). Data collected from 12 years of the ICS quality control program for S. pneumoniae demonstrate excellent (≥95%) overall concordance for serotype and antimicrobial susceptibility testing results across six laboratories. IMPORTANCE: Arctic populations experience several social and physical challenges that lead to the increased spread and incidence of invasive diseases. The International Circumpolar Surveillance (ICS) program was developed to monitor five invasive bacterial diseases in Arctic countries and territories. Each ICS organism has a corresponding interlaboratory quality control (QC) program for laboratory-based typing, to ensure the technical precision and accuracy of reference testing services for these regions, and identify and correct potential problems. Here, we describe the results of the ICS Streptococcus pneumoniae QC program, from 2009 to 2020. Excellent overall concordance was achieved for serotype and antimicrobial susceptibility testing results across six laboratories. Ongoing participation in these QC programs ensures the continuation of quality surveillance systems within Arctic populations that experience health disparities.

Serotype distribution, virulence and antibiotic resistance of Streptococcus agalactiae isolated from cultured tilapia Oreochromis niloticus in Lake Volta, Ghana.

Streptococcus agalactiae infection is one of the major factors limiting the expansion of tilapia farming globally. In this study, we investigated the serotype distribution, virulence and antimicrobial resistance of S. agalactiae isolates from tilapia farmed in Lake Volta, Ghana. Isolates from 300 moribund fish were characterised by Gram staining, MALDI-TOF/MS and 16S rRNA sequencing. Serotype identification was based on multiplex polymerase chain reaction (PCR) amplification of the capsular polysaccharide genes. Detection of virulence genes (cfb, fbsA and cspA) and histopathology were used to infer the pathogenicity of the isolates. The susceptibility of isolates to antibiotics was tested using the Kirby-Bauer disk diffusion assay. All 32 isolates identified as S. agalactiae were of serotype Ia. This was notably different from isolates previously collected from the farms in 2017, which belonged to serotype Ib, suggesting a possible serotype replacement. The prevalence of the pathogen was related to the scale of farm operation, with large-scale farms showing higher S. agalactiae positivity. Data from histopathological analysis and PCR amplification of targeted virulence genes confirmed the virulence potential and ability of the isolates to cause systemic infection in tilapia. Except for gentamicin, the majority of the isolates were less resistant to the tested antibiotics. All isolates were fully sensitive to oxytetracycline, erythromycin, florfenicol, enrofloxacin, ampicillin and amoxicillin. This study has improved our understanding of the specific S. agalactiae serotypes circulating in Lake Volta and demonstrates the need for continuous monitoring to guide the use of antimicrobials and vaccines against streptococcal infections in Ghanaian aquaculture systems.

Serotype diversity and antimicrobial susceptibility profiles of Actinobacillus pleuropneumoniae isolated in Italian pig farms from 2015 to 2022.

Actinobacillus pleuropneumoniae (APP) is a bacterium frequently associated with porcine pleuropneumonia. The acute form of the disease is highly contagious and often fatal, resulting in significant economic losses for pig farmers. Serotype diversity and antimicrobial resistance (AMR) of APP strains circulating in north Italian farms from 2015 to 2022 were evaluated retrospectively to investigate APP epidemiology in the area. A total of 572 strains isolated from outbreaks occurring in 337 different swine farms were analysed. The majority of isolates belonged to serotypes 9/11 (39.2%) and 2 (28.1%) and serotype diversity increased during the study period, up to nine different serotypes isolated in 2022. The most common resistances were against tetracycline (53% of isolates) and ampicillin (33%), followed by enrofloxacin, florfenicol and trimethoprim/sulfamethoxazole (23% each). Multidrug resistance (MDR) was common, with a third of isolates showing resistance to more than three antimicrobial classes. Resistance to the different classes and MDR varied significantly depending on the serotype. In particular, the widespread serotype 9/11 was strongly associated with florfenicol and enrofloxacin resistance and showed the highest proportion of MDR isolates. Serotype 5, although less common, showed instead a concerning proportion of trimethoprim/sulfamethoxazole resistance. Our results highlight how the typing of circulating serotypes and the analysis of their antimicrobial susceptibility profile are crucial to effectively manage APP infection and improve antimicrobial stewardship.

The use of antigens derived from Bacillus thuringiensis bacteria for further differentiation.

This study is devoted to studying Bacillus thuringiensis antigens and their insecticide activity as critical features in bacterial differentiation. Indeed, 190 samples were examined for flagellar antigenicity as well as the insecticidal activity exhibited. From a serological perspective, 122 isolates (64.2 %) were attributed to 8 H-serogroups, including 3 non-typeable and 65 unverified. The dominant serotype was H3abc (82 % frequency); H6 was less frequent (8.5 %). The other 6 serotypes accounted for a low frequency of occurrence (up to 1.5 %). Of the 190 isolates tested, 125 (65.8 %) formed bipyramidal, and 63 (33.2 %) represented spherical inclusions. All H3abc isolates contained bipyramidal inclusions. The same applied to H8ab and H7 isolates. Insecticide activity was noted in 70.1 % of the population. In general, 128 samples were toxic to both species (Bombyx mori, Aedes sp.). Another 3 samples were toxic only to B. mori, and 2 for Aedes sp. Among the samples exhibiting toxicity to both species, 97.6 % belonged to bipyramidal paraspore inclusions (H3abc). All H7 samples were toxic to two insect species. Monotoxic B. thuringiensis against Aedes sp. were found only among organisms producing spherical parasporal inclusions in the cell. Examples of such microorganisms include an isolate of the H4ab/43 serotype.

A Combined Approach for the Characterization of Small Ruminant Lentivirus Strains Circulating in the Islands and Mainland of Greece.

Small ruminant lentiviruses are a group of viruses infecting goat and sheep worldwide. These viruses exhibit an extraordinary degree of genetic and antigenic variability that severely influence in vivo and in vitro features, as well as diagnostic test results. Small ruminant farming is the most important animal farming business in Greece, with a high impact on the Greek primary economy. Although SRLV infection and its impact on animal production are well established in the country, little is known about the circulating SRLV strains and their prevalence. The aim of this study was to characterize SRLVs circulating in Greece with a combined serological and molecular approach, using the bulk milk matrix collected from 60 farms in different municipalities. This study allowed us to estimate a seroprevalence of around 52% at the herd level. The B1, B2 and A3 subtypes and a novel A viral cluster were identified. Moreover, the amplicon sequencing method allowed us to identify more than one viral subtype in a sample. These results again confirm the high variability of these viruses and highlight the importance of the constant monitoring of viral evolution, in particular in antigens of diagnostic interest.

Shigella Detection and Molecular Serotyping With a Customized TaqMan Array Card in the Enterics for Global Health (EFGH): Shigella Surveillance Study.

Background: Quantitative polymerase chain reaction (qPCR) targeting ipaH has been proven to be highly efficient in detecting Shigella in clinical samples compared to culture-based methods, which underestimate Shigella burden by 2- to 3-fold. qPCR assays have also been developed for Shigella speciation and serotyping, which is critical for both vaccine development and evaluation. Methods: The Enterics for Global Health (EFGH) Shigella surveillance study will utilize a customized real-time PCR-based TaqMan Array Card (TAC) interrogating 82 targets, for the detection and differentiation of Shigella spp, Shigella sonnei, Shigella flexneri serotypes, other diarrhea-associated enteropathogens, and antimicrobial resistance (AMR) genes. Total nucleic acid will be extracted from rectal swabs or stool samples, and assayed on TAC. Quantitative analysis will be performed to determine the likely attribution of Shigella and other particular etiologies of diarrhea using the quantification cycle cutoffs derived from previous studies. The qPCR results will be compared to conventional culture, serotyping, and phenotypic susceptibility approaches in EFGH. Conclusions: TAC enables simultaneous detection of diarrheal etiologies, the principal pathogen subtypes, and AMR genes. The high sensitivity of the assay enables more accurate estimation of Shigella-attributed disease burden, which is critical to informing policy and in the design of future clinical trials.

Targeted Next-Generation Sequencing Assay for Direct Detection and Serotyping of Salmonella from Enrichment.

In this study, an automated, targeted next-generation sequencing (tNGS) assay to detect and serotype Salmonella from sample enrichments was evaluated. The assay generates millions of reads to detect multiple Salmonella-specific genes and serotype-specific alleles, detecting all Salmonella spp. tested to date, and serotyping 62 common Salmonella serotypes. Accuracy was tested on 291 pure reference cultures (251 Salmonella, 40 non-Salmonella), 21 artificially contaminated poultry carcass rinse samples, and 363 naturally contaminated poultry environmental samples. Among the 291 pure reference cultures, the automated tNGS assay resulted in 100% detection accuracy, 100% serotyping accuracy for the claimed serotypes, and 0% false positives. The limit of detection was estimated at 5 × 104 CFU/mL by testing enumerated cultures of strains representative of six serotypes. In cocontamination studies with mixtures of two serotypes (Enteritidis, Typhimurium, Kentucky, Infantis, and Newport) at a 1:1 ratio, tNGS detected both serotypes with 100% accuracy. The assay demonstrated 100% accuracy in artificially contaminated poultry carcass rinse sample enrichments. Targeted NGS was highly effective in detecting Salmonella in samples collected from poultry production facilities. Results demonstrated that tNGS could detect Salmonella and provide accurate serotyping information consistent with conventional serology. These findings highlight the reliable and efficient performance of a fully automated tNGS Salmonella assay in detecting and identifying Salmonella strains in complex matrices, reducing the time to results from 4 to 5 days required by the traditional isolation and serotyping to 10-12 h for tNGS after primary enrichment.

Macrolide and lincosamide resistance of Streptococcus agalactiae in pregnant women in Poland.

Knowing about the antibiotic resistance, serotypes, and virulence-associated genes of Group B Streptococcus for epidemiological and vaccine development is very important. We have determined antimicrobial susceptibility patterns, serotype, and virulence profiles. The antibiotic susceptibility was assessed for a total of 421 Streptococcus agalactiae strains, isolated from pregnant women and neonates. Then, 89 erythromycin and/or clindamycin-resistant strains (82 isolates obtained from pregnant women and seven isolates derived from neonates) were assessed in detail. PCR techniques were used to identify the studied strains, perform serotyping, and assess genes encoding selected virulence factors. Phenotypic and genotypic methods determined the mechanisms of resistance. All tested strains were sensitive to penicillin and levofloxacin. The constitutive MLSB mechanism (78.2%), inducible MLSB mechanism (14.9%), and M phenotype (6.9%) were identified in the macrolide-resistant strains. It was found that macrolide resistance is strongly associated with the presence of the ermB gene and serotype V. FbsA, fbsB, fbsC, scpB, and lmb formed the most recurring pattern of genes among the nine surface proteins whose genes were analysed. A minority (7.9%) of the GBS isolates exhibited resistance to lincosamides and macrolides, or either, including those that comprised the hypervirulent clone ST-17. The representative antibiotic resistance pattern consisted of erythromycin, clindamycin, and tetracycline resistance (71.9%). An increase in the fraction of strains resistant to macrolides and lincosamides indicates the need for monitoring both the susceptibility of these strains and the presence of the ST-17 clone.

Rare serotype c Haemophilus influenzae invasive isolate: characterization of the first case in Portugal.

We report for the first time in Portugal a serotype c Haemophilus influenzae isolated from an adult, with HIV-1 infection. Whole-genome sequencing characterized the isolate as clonal complex ST-7, albeit with a novel MLST (ST2754) due to a unique atpG profile. Integration of this genome with other available H. influenzae serotype c genomes from PubMLST revealed its overall genetic distinctiveness, with the closest related isolate being identified in France in 2020. This surveillance study, involving collaboration among hospitals and reference laboratory, successfully contributed to the identification and characterization of this rare serotype.

Antimicrobial Resistance of Streptococcus pneumoniae Clinical Serotypes between 2017 and 2022 in Crete, Greece.

BACKGROUND: Pneumococcal disease is still considered a global problem. With the introduction of pneumococcal conjugate vaccines (PCVs) serotype epidemiology changed, but antimicrobial resistance persists constituting a serious problem. The current study aimed to determine the serotype distribution and the antimicrobial susceptibility of recent Streptococcus pneumoniae isolates, following implementation of the 13-valent conjugate vaccine (PCV13). MATERIALS AND METHODS: From January 2017 to December 2022 we evaluated 116 nonduplicate S. pneumoniae isolates collected from adult patients (21 - 98 years) cared for in the University Hospital of Heraklion, Crete, Greece. Pneumococcal isolates were serotyped by the Quellung reaction, and antimicrobial susceptibility testing was performed using E-test. Multidrug resistance (MDR) was defined as non-susceptibility to at least one agent in ≥3 classes of antibiotics. RESULTS: Among the 116 isolates, 31% were recognized as invasive pneumococcal strains, while 69% were non-invasive. The isolates tested belonged to 25 different serotypes. The most prevalent serotypes were 11A (10.3%), and 35B (10.3%), followed by 3 (9.5%), 15A (7.8%), 25F (6.9%), 19A (5.3%), 35F (5.3%), and others (44.6%). The coverage rates of PCV13 and the pneumococcal polysaccharide vaccine (PPSV23) were 26.7% and 57.8%, respectively. PCV13 and PPSV23 serotypes decreased between 2017 - 2019 and 2020 - 2022, with a parallel increase in the non-vaccine types. Resistance rates to erythromycin, clindamycin, trimethoprim/sulfamethoxazole, penicillin, levofloxacin, and ceftriaxone, were 40.5%, 21.6%, 13.8%, 12.1%, 3.4%, and 0%, respectively. All isolates were susceptible to vancomycin, linezolid, and daptomycin. MDR was observed among 36 (31%) S. pneumoniae isolates. CONCLUSION: The increasing levels of resistance in S. pneumoniae in Crete, Greece, highlight the need for continuous surveillance of antimicrobial resistance and development of strategies for its reduction, including antimicrobial stewardship programs, increased pneumococcal vaccination, and development of next generation PCVs with a wider serotype coverage.