Distribution of Chlamydia trachomatis ompA-genotypes over three decades in Portugal.

OBJECTIVES: Chlamydia trachomatis is classified into 15 major genotypes, A to L3, based on the diversity of ompA gene. Here, we evaluated and characterised the distribution and diversity of ompA-genotypes over 32 years (1990-2021) in Portugal. METHODS: The collection of the Portuguese National Reference Laboratory for Sexually Transmitted Infections includes 5824 C. trachomatis-positive samples that were successfully ompA-genotyped between 1990 and 2021. An in-depth analysis of ompA-genotypes distribution across the years, as well as by biological sex, age and anatomical site of infection was performed. RESULTS: ompA-genotype E was consistently the most frequently detected across the years, with a median frequency of 34.6%, followed by D/Da (17.6%), F (14.3%) and G (10.7%). The prevalence of lymphogranuloma venereum (LGV) genotypes (mostly L2, 62.0%, followed by L2b, 32.1%) increased since 2016, reaching the highest value in 2019 (20.9%). LGV, G and Da genotypes were associated with biological sex, specifically with being male, and were the most frequent among anorectal specimens (37.7%, 19.4% and 17.7%, respectively). Notably, LGV ompA-genotypes represented 38.9% of the male anorectal specimens since 2016, and were also detected among oropharynx and urogenital samples. ompA-genotype E was the most frequently detected at the oropharynx (28.6%) and urogenital (33.9%) sites during the study period, followed by D/Da (17.4%) and F (16.0%) in the urogenital specimens, and by G (26.1%) and D/Da (25.7%) in oropharynx specimens. Our data also highlight the emergence of the recombinant L2b/D-Da strain since 2017 (representing between 2.0% and 15.5% of LGV cases per year) and the non-negligible detection of ompA-genotype B in urogenital and anorectal specimens. CONCLUSIONS: This study provides a comprehensive landscape of C. trachomatis molecular surveillance in Portugal, highlighting the continued relevance of ompA-genotyping as a complement to rapid LGV-specific detection tests. It also contributes to a deeper understanding of C. trachomatis epidemiology, diversity and pathogenicity.

The influence of nutrition in nodular thyroid pathology: a systematic review.

Nodular thyroid pathologies (NThyPs) are prevalent thyroid diseases, with a steadily increasing global incidence. Although their exact causes remain uncertain, various modifying factors, such as nutrition, influence their development. We aimed to systematically identify and synthesize the influence of dietary exposures on NThyPs risk. PubMed, Scopus, Web of Science, and EMBASE were searched on June 14, 2024, to identify relevant studies. Data extraction included study characteristics, sociodemographic factors, dietary intake assessments and exposures, and NThyPs subtypes. Out of 14,730 articles retrieved, 55 observational or experimental studies, in English, Spanish, or Portuguese, that assessed nutrition's impact on NThyPs were included, excluding non-human studies, reviews, meta-analyses, and publications outside the study's scope. Forty studies investigated how distinct dietary patterns, macronutrients, minerals, and vitamins contributed to or mitigated NThyPs development. Fish and seafood, vegetables and fruits, and meat were other dietary exposures investigated. In fourteen of previously selected studies, drink consumption was also addressed. This systematic review (CRD420234003439) suggests that reducing processed foods, sugars, meat, and dietary iodine lowers NThyPs risk, while coffee, tea, alcohol, and dairy products demonstrates potential protective roles. Fish and seafood, and fruits and vegetables also exhibit protective properties; nevertheless, further research is necessary to establish definitive conclusions.

Comparative Effectiveness of COVID-19 Vaccines in Preventing Infections and Disease Progression from SARS-CoV-2 Omicron BA.5 and BA.2, Portugal.

We estimated comparative primary and booster vaccine effectiveness (VE) of SARS-CoV-2 Omicron BA.5 and BA.2 lineages against infection and disease progression. During April-June 2022, we implemented a case-case and cohort study and classified lineages using whole-genome sequencing or spike gene target failure. For the case-case study, we estimated the adjusted odds ratios (aORs) of vaccination using a logistic regression. For the cohort study, we estimated VE against disease progression using a penalized logistic regression. We observed no reduced VE for primary (aOR 1.07 [95% CI 0.93-1.23]) or booster (aOR 0.96 [95% CI 0.84-1.09]) vaccination against BA.5 infection. Among BA.5 case-patients, booster VE against progression to hospitalization was lower than that among BA.2 case-patients (VE 77% [95% CI 49%-90%] vs. VE 93% [95% CI 86%-97%]). Although booster vaccination is less effective against BA.5 than against BA.2, it offers substantial protection against progression from BA.5 infection to severe disease.

Epidemiology and genetic diversity of invasive Neisseria meningitidis strains circulating in Portugal from 2003 to 2020.

Invasive meningococcal disease (IMD) continues to be a public health problem due to its epidemic potential, affecting mostly children. We aimed to present a detailed description of the epidemiology of IMD in Portugal, including insights into the genetic diversity of Neisseria meningitidis strains. Epidemiological analysis included data from the Portuguese National Reference Laboratory of Neisseria meningitidis during 2003 to 2020. Since 2012, N. meningitidis isolates have also been assessed for their susceptibility to antibiotics and were characterized by whole genome sequencing. During 2003-2020, 1392 confirmed cases of IMD were analyzed. A decrease in the annual incidence rate was observed, ranging from 1.99 (2003) to 0.39 (2020), with an average case fatality rate of 7.1%. Serogroup B was the most frequent (69.7%), followed by serogroups C (9.7%), Y (5.7%), and W (2.6%). Genomic characterization of 329 isolates identified 20 clonal complexes (cc), with the most prevalent belonging to serogroup B cc41/44 (26.3%) and cc213 (16.3%). Isolates belonging to cc11 were predominantly from serogroups W (77.3%) and C (76.5%), whereas cc23 was dominant from serogroup Y (65.7%). Over the past 4 years (2017-2020), we observed an increasing trend of cases assigned to cc213, cc32, and cc11. Regarding antimicrobial susceptibility, all isolates were susceptible to ceftriaxone and 61.8% were penicillin-nonsusceptible, whereas 1.4% and 1.0% were resistant to ciprofloxacin and rifampicin. This is the first detailed study on the epidemiology and genomics of invasive N. meningitidis infections in Portugal, providing relevant data to public health policy makers for a more effective control of this disease.

Distribution and Clinical Significance of HPV16 Variants in Head and Neck Squamous Cell Carcinomas: Data from a Portuguese Cohort and Systematic Review.

INTRODUCTION: Genomic variants of the human papillomavirus type 16 (HPV16) are thought to play differential roles in the susceptibility to head and neck squamous cell carcinomas (HNSCC) and its biological behaviour. This study aimed to establish the prevalence of HPV16 variants in an HNSCC cohort and associate them with clinical pathological characteristics and patient survival. METHODS: We retrieved samples and clinical data from 68 HNSCC patients. DNA samples were available from tumour biopsy at the time of the primary diagnosis. Targeted next-generation sequencing was used to obtain whole-genome sequences, and variants were established based on phylogenetic classification. RESULTS: 74% of samples clustered in lineage A, 5.7% in lineage B, 2.9% in lineage C, and 17.1% in lineage D. Comparative genome analysis revealed 243 single nucleotide variations. Of these, one hundred were previously reported, according to our systematic review. No significant associations with clinical pathological variables or patient survival were observed. The E6 amino acid variations E31G, L83V, and D25E and E7 N29S, associated with cervical cancer, were not observed, except for N29S in a single patient. CONCLUSION: These results provide a comprehensive genomic map of HPV16 in HSNCC, highlighting tissue-specific characteristics which will help design tailored therapies for cancer patients.

Genomic surveillance of Neisseria meningitidis serogroup W in Portugal from 2003 to 2019.

In recent years, a change in the epidemiology of meningococcal disease caused by Neisseria meningitidis serogroup W (MenW) has been observed worldwide, with the emergence of new sublineages associated with a higher rate of fatal cases. The present study intends to describe the epidemiology of invasive meningococcal disease (IMD) due to MenW in Portugal between 2003 and 2019, and to genetically characterize population structure. Despite MenW has a low incidence in Portugal, having almost disappeared from 2008 to 2015, since 2016, the number of MenW cases has been steadily increasing at a rate of ~ twofold per year, with more than 80% of the characterized isolates belonging to clonal complex 11 (cc11). Core-genome phylogeny of 25 Portuguese (PT) MenW isolates showed a strain clustering mainly either with the Original UK or the UK 2013 sublineages. Our study also reported for the first time the presence of distinct prophages with a notable overrepresentation of an ~ 32-35-kb PS_1-like prophage found in MenW cc11 genomes. The presence of the PS_1-like prophage in almost all 4723 cc11 genomes selected from Neisseria PubMLST database regardless of the capsular group they belong to suggests an ancestral acquisition of this mobile element prior to capsular switching events. Overall, by mimicking the scenario observed worldwide, this study reinforces the importance of a close monitoring of MenW disease, especially from cc11, in order to promptly adapt the vaccination plan for IMD control in Portugal. Moreover, future studies are needed to understand the putative contribution of prophages to fitness and virulence of PT MenW strains.

Global gene expression analysis of Streptococcus agalactiae at exponential growth phase.

A comparative overview of the global gene expression levels of S. agalactiae reference strain NEM316 at the exponential growth phase was done through RNA-sequencing. The expression levels of 47 genes potentially linked to virulence evidenced that: i) the major nuclease, GBS_RS03720/gbs0661, presented higher mean expression values than the remainder of DNase genes; ii) the genetic pilus island PI-2a genes presented higher mean expression values than PI-1 coding genes; and, iii) three virulence-associated genes ranked among the top-100 most expressed genes (GBS_RS07760, GBS_RS09445 and GBS_RS03485). Among this top-100, genes encoding proteins involved in "Translation, ribosomal structure and biogenesis" represented 46%. Curiously, genes with no assigned function were grouped in the category of highly expressed genes. As very little is known about the molecular mechanisms behind the release of DNases, preliminary assays were developed to understand whether direct DNA exposure would affect gene expression at the exponential growth phase. No differentially expressed genes were detected, indicating that follow-up studies are needed to disclose the complex molecular pathways (and stimuli) triggering the release of DNases. In general, our insights on the global expression levels of NEM316 at exponential growth phase with and without DNA exposure should open novel research lines to decipher S. agalactiae puzzling adaptation and virulence mechanisms, such as DNase production.

Airborne spores' dissemination of a swine associated Clostridioides difficile clone.

This study supports the airborne dissemination of Clostridioides difficile spores. Of the sieve impaction samples collected at a swine production unit, 66.7% were positive and all belonged to the predominantly established clone. Spores' density varied according to the characteristics of the animal population, suggesting the possibility of airborne transmission.

The Long-Term Culture of Human Fibroblasts Reveals a Spectroscopic Signature of Senescence.

Aging is a complex process which leads to progressive loss of fitness/capability/ability, increasing susceptibility to disease and, ultimately, death. Regardless of the organism, there are some features common to aging, namely, the loss of proteostasis and cell senescence. Mammalian cell lines have been used as models to study the aging process, in particular, cell senescence. Thus, the aim of this study was to characterize the senescence-associated metabolic profile of a long-term culture of human fibroblasts using Fourier Transform Infrared and Nuclear Magnetic Resonance spectroscopy. We sub-cultivated fibroblasts from a newborn donor from passage 4 to passage 17 and the results showed deep changes in the spectroscopic profile of cells over time. Late passage cells were characterized by a decrease in the length of fatty acid chains, triglycerides and cholesterol and an increase in lipid unsaturation. We also found an increase in the content of intermolecular ß-sheets, possibly indicating an increase in protein aggregation levels in cells of later passages. Metabolic profiling by NMR showed increased levels of extracellular lactate, phosphocholine and glycine in cells at later passages. This study suggests that spectroscopy approaches can be successfully used to study changes concomitant with cell senescence and validate the use of human fibroblasts as a model to monitor the aging process.

Characterization of the Human Papillomavirus 16 Oncogenes in K14HPV16 Mice: Sublineage A1 Drives Multi-Organ Carcinogenesis.

The study of human papillomavirus (HPV)-induced carcinogenesis uses multiple in vivo mouse models, one of which relies on the cytokeratin 14 gene promoter to drive the expression of all HPV early oncogenes. This study aimed to determine the HPV16 variant and sublineage present in the K14HPV16 mouse model. This information can be considered of great importance to further enhance this K14HPV16 model as an essential research tool and optimize its use for basic and translational studies. Our study evaluated HPV DNA from 17 samples isolated from 4 animals, both wild-type (n = 2) and HPV16-transgenic mice (n = 2). Total DNA was extracted from tissues and the detection of HPV16 was performed using a qPCR multiplex. HPV16-positive samples were subsequently whole-genome sequenced by next-generation sequencing techniques. The phylogenetic positioning clearly shows K14HPV16 samples clustering together in the sub-lineage A1 (NC001526.4). A comparative genome analysis of K14HPV16 samples revealed three mutations to the human papillomaviruses type 16 sublineage A1 representative strain. Knowledge of the HPV 16 variant is fundamental, and these findings will allow the rational use of this animal model to explore the role of the A1 sublineage in HPV-driven cancer.

FTIR Spectroscopy as a Tool to Study Age-Related Changes in Cardiac and Skeletal Muscle of Female C57BL/6J Mice.

Studying aging is important to further understand the molecular mechanisms underlying this physiological process and, ideally, to identify a panel of aging biomarkers. Animals, in particular mice, are often used in aging studies, since they mimic important features of human aging, age quickly, and are easy to manipulate. The present work describes the use of Fourier Transform Infrared (FTIR) spectroscopy to identify an age-related spectroscopic profile of the cardiac and skeletal muscle tissues of C57BL/6J female mice. We acquired ATR-FTIR spectra of cardiac and skeletal muscle at four different ages: 6; 12; 17 and 24 months (10 samples at each age) and analyzed the data using multivariate statistical tools (PCA and PLS) and peak intensity analyses. The results suggest deep changes in protein secondary structure in 24-month-old mice compared to both tissues in 6-month-old mice. Oligomeric structures decreased with age in both tissues, while intermolecular ß-sheet structures increased with aging in cardiac muscle but not in skeletal muscle. Despite FTIR spectroscopy being unable to identify the proteins responsible for these conformational changes, this study gives insights into the potential of FTIR to monitor the aging process and identify an age-specific spectroscopic signature.

Lung abscess due to Neisseria meningitidis serogroup X-unexpected virulence of a commensal resulting from putative serogroup B capsular switching.

To report the first case of a lung abscess caused by Neisseria meningitidis (Nm) and to genetically characterize the rare underlying capsule switching event. The strain (PT NmX) was subjected to whole genome sequencing, and a comparative gene-by-gene analysis was performed based on 1605 N. meningitidis core loci that constitute the MLST core-genome scheme (cgMLST) V1.0. All ~ 9,600 genomes available on Neisseria PubMLST (until 30th November 2019) from all serogroups were used to better identify the genome make-up of the PT NmX strain. This strain was found to be highly divergent from other NmX reported worldwide and to belong to a new sequence type (ST-14273), with the finetype X: P1.19,15-1:F5-2. Moreover, it revealed a closer genetic proximity to strains from serogroup B than to other serogroups, suggesting a genome backbone associated with serogroup B, while it presents a capsule synthesis region derived from a NmX strain. We describe a new hybrid NmB/X isolate from a noninvasive meningococcal infection, causing lung abscess. Despite capsular switching events involving serogroup X are rare, it may lead to the emergence of pathogenic potential. Studies should continue to better understand the molecular basis underlying Neisseria strains' ability to spread to body compartments other than the tissues for which their tropism is already known.

Proficiency Testing of Virus Diagnostics Based on Bioinformatics Analysis of Simulated In Silico High-Throughput Sequencing Data Sets.

Quality management and independent assessment of high-throughput sequencing-based virus diagnostics have not yet been established as a mandatory approach for ensuring comparable results. The sensitivity and specificity of viral high-throughput sequence data analysis are highly affected by bioinformatics processing using publicly available and custom tools and databases and thus differ widely between individuals and institutions. Here we present the results of the COMPARE [Collaborative Management Platform for Detection and Analyses of (Re-)emerging and Foodborne Outbreaks in Europe] in silico virus proficiency test. An artificial, simulated in silico data set of Illumina HiSeq sequences was provided to 13 different European institutes for bioinformatics analysis to identify viral pathogens in high-throughput sequence data. Comparison of the participants' analyses shows that the use of different tools, programs, and databases for bioinformatics analyses can impact the correct identification of viral sequences from a simple data set. The identification of slightly mutated and highly divergent virus genomes has been shown to be most challenging. Furthermore, the interpretation of the results, together with a fictitious case report, by the participants showed that in addition to the bioinformatics analysis, the virological evaluation of the results can be important in clinical settings. External quality assessment and proficiency testing should become an important part of validating high-throughput sequencing-based virus diagnostics and could improve the harmonization, comparability, and reproducibility of results. There is a need for the establishment of international proficiency testing, like that established for conventional laboratory tests such as PCR, for bioinformatics pipelines and the interpretation of such results.

Imipenem Resistance in Clostridium difficile Ribotype 017, Portugal.

We describe imipenem-resistant and imipenem-susceptible clinical isolates of Clostridium difficile ribotype 017 in Portugal. All ribotype 017 isolates carried an extra penicillin-binding protein gene, pbp5, and the imipenem-resistant isolates had additional substitutions near the transpeptidase active sites of pbp1 and pbp3. These clones could disseminate and contribute to imipenem resistance.

Whole-Genome Sequence Analysis of Multidrug-Resistant Campylobacter Isolates: a Focus on Aminoglycoside Resistance Determinants.

A whole-genome sequencing (WGS) approach was conducted in order to identify the molecular determinants associated with antimicrobial resistance in 12 multidrug-resistant Campylobacter jejuni and Campylobacter coli isolates, with a focus on aminoglycoside resistance determinants. Two variants of a new aminoglycoside phosphotransferase gene [aph(2″)-Ii1 and aph(2″)-Ii2 ] putatively associated with gentamicin resistance were found. In addition, the following new genes were identified for the first time in Campylobacter: a lincosamide nucleotidyltransferase gene [lnu(G)], likely associated with lincomycin resistance, and two resistance enzyme genes (spw and apmA) similar to those found in Staphylococcus aureus, which may confer spectinomycin and gentamicin resistance, respectively. A C1192T mutation of the 16S rRNA gene that may be involved in spectinomycin resistance was also found in a C. coli isolate. Genes identified in the present study were located either on the bacterial chromosome or on plasmids that could be transferred naturally. Their role in aminoglycoside resistance remains to be supported by genetic studies. Regarding the other antimicrobial agents studied, i.e., ampicillin, ciprofloxacin, erythromycin, and tetracycline, a perfect correlation between antimicrobial phenotypes and genotypes was found. Overall, our data suggest that WGS analysis is a powerful tool for identifying resistance determinants in Campylobacter and can disclose the full genetic elements associated with resistance, including antimicrobial compounds not tested routinely in antimicrobial susceptibility testing.

The expression of Helicobacter pylori tfs plasticity zone cluster is regulated by pH and adherence, and its composition is associated with differential gastric IL-8 secretion.

BACKGROUND: Helicobacter pylori virulence is associated with different clinical outcomes. The existence of an intact dupA gene from tfs4b cluster has been suggested as a predictor for duodenal ulcer development. However, the role of tfs plasticity zone clusters in the development of ulcers remains unclear. We studied several H. pylori strains to characterize the gene arrangement of tfs3 and tfs4 clusters and their impact in the inflammatory response by infected gastric cells. METHODS: The genome of 14 H. pylori strains isolated from Western patients, pediatric (n=10) and adult (n=4), was fully sequenced using the Illumina platform MiSeq, in addition to eight pediatric strains previously sequenced. These strains were used to infect human gastric cells, and the secreted interleukin-8 (IL-8) was quantified by ELISA. The expression of virB2, dupA, virB8, virB10, and virB6 was assessed by quantitative PCR in adherent and nonadherent fractions of H. pylori during in vitro co-infection, at different pH values. RESULTS: We have found that cagA-positive H. pylori strains harboring a complete tfs plasticity zone cluster significantly induce increased production of IL-8 from gastric cells. We have also found that the region spanning from virB2 to virB10 genes constitutes an operon, whose expression is increased in the adherent fraction of bacteria during infection, as well as in both adherent and nonadherent fractions at acidic conditions. CONCLUSIONS: A complete tfs plasticity zone cluster is a virulence factor that may be important for the colonization of H. pylori and to the development of severe outcomes of the infection with cagA-positive strains.

In Vitro Activity of Quaternary Ammonium Surfactants against Streptococcal, Chlamydial, and Gonococcal Infective Agents.

Quaternary ammonium compounds (QAC) are widely used, cheap, and chemically stable disinfectants and topical antiseptics with wide-spectrum antimicrobial activities. Within this group of compounds, we recently showed that there are significant differences between the pharmacodynamics of n-alkyl quaternary ammonium surfactants (QAS) with a short (C12) alkyl chain when in vitro toxicities toward bacterial and mammalian epithelial cells are compared. These differences result in an attractive therapeutic window that justifies studying short-chain QAS as prophylactics for sexually transmitted infections (STI) and perinatal vertically transmitted urogenital infections (UGI). We have evaluated the antimicrobial activities of short-chain (C12) n-alkyl QAS against several STI and UGI pathogens as well as against commensal Lactobacillus species. Inhibition of infection of HeLa cells by Neisseria gonorrhoeae and Chlamydia trachomatis was studied at concentrations that were not toxic to the HeLa cells. We show that the pathogenic bacteria are much more susceptible to QAS toxic effects than the commensal vaginal flora and that QAS significantly attenuate the infectivity of N. gonorrhoeae and C. trachomatis without affecting the viability of epithelial cells of the vaginal mucosa. N-Dodecylpyridinium bromide (C12PB) was found to be the most effective QAS. Our results strongly suggest that short-chain (C12) n-alkyl pyridinium bromides and structurally similar compounds are promising microbicide candidates for topical application in the prophylaxis of STI and perinatal vertical transmission of UGI.

Quaternary ammonium surfactant structure determines selective toxicity towards bacteria: mechanisms of action and clinical implications in antibacterial prophylaxis.

OBJECTIVES: Broad-spectrum antimicrobial activity of quaternary ammonium surfactants (QAS) makes them attractive and cheap topical prophylactic options for sexually transmitted infections and perinatal vertically transmitted urogenital infections. Although attributed to their high affinity for biological membranes, the mechanisms behind QAS microbicidal activity are not fully understood. We evaluated how QAS structure affects antimicrobial activity and whether this can be exploited for use in prophylaxis of bacterial infections. METHODS: Acute toxicity of QAS to in vitro models of human epithelial cells and bacteria were compared to identify selective and potent bactericidal agents. Bacterial cell viability, membrane integrity, cell cycle and metabolism were evaluated to establish the mechanisms involved in selective toxicity of QAS. RESULTS: QAS toxicity normalized relative to surfactant critical micelle concentration showed n-dodecylpyridinium bromide (C12PB) to be the most effective, with a therapeutic index of ∼10 for an MDR strain of Escherichia coli and >20 for Neisseria gonorrhoeae after 1 h of exposure. Three modes of QAS antibacterial action were identified: impairment of bacterial energetics and cell division at low concentrations; membrane permeabilization and electron transport inhibition at intermediate doses; and disruption of bacterial membranes and cell lysis at concentrations close to the critical micelle concentration. In contrast, toxicity to mammalian cells occurs at higher concentrations and, as we previously reported, results primarily from mitochondrial dysfunction and apoptotic cell death. CONCLUSIONS: Our data show that short chain (C12) n-alkyl pyridinium bromides have a sufficiently large therapeutic window to be good microbicide candidates.

Role of inter-species recombination of the ftsI gene in the dissemination of altered penicillin-binding-protein-3-mediated resistance in Haemophilus influenzae and Haemophilus haemolyticus.

OBJECTIVES: To screen the ftsI gene sequences obtained from clinical isolates of non-typeable Haemophilus influenzae (NTHi) and Haemophilus haemolyticus for the presence of mosaic ftsI gene structures, and to evaluate the role of inter-species recombination of the ftsI gene in the formation and distribution of resistant ftsI genes. METHODS: The ftsI genes of 100 Haemophilus isolates comprising genetically defined ß-lactamase-negative ampicillin-susceptible (gBLNAS), ß-lactamase-positive ampicillin-resistant (gBLPAR), ß-lactamase-negative ampicillin-resistant (gBLNAR) and ß-lactamase-positive amoxicillin/clavulanate-resistant (gBLPACR) isolates of NTHi (n = 50) and H. haemolyticus (n = 50) were analysed in this study. Both the flanking regions and the full-length ftsI gene sequences of all study isolates were screened for mosaic structures using H. influenzae Rd and H. haemolyticus ATCC 33390 as reference parental sequences, and bioinformatics methods were used for recombination analysis using SimPlot. RESULTS: Of the 100 clinical isolates analysed 34% (34/100) harboured mosaic ftsI gene structures containing distinct ftsI gene fragments similar to both reference parental sequences. The inter-species recombination events were exclusively encountered in the ftsI gene of gBLNAR/gBLPACR isolates of both NTHi and H. haemolyticus, and were always associated with the formation of a mosaic fragment at the 3' end of the ftsI gene. There was no evidence supporting horizontal gene transfer (HGT) involving the entire ftsI gene among the clinical isolates in vivo. CONCLUSIONS: We provide evidence for the HGT and inter-species recombination of the ftsI gene among gBLNAR/gBLPACR isolates of NTHi and H. haemolyticus in a clinical setting, highlighting the importance of recombination of the ftsI gene in the emergence of altered penicillin-binding protein 3 and BLNAR-mediated resistance.