Detection of CTX-M-15 ESBL in XDR Haemophilus parainfluenzae from a urethral swab.

OBJECTIVES: Haemophilus parainfluenzae is an opportunistic pathogen causing respiratory tract infection and sexually transmitted diseases. The emergence of multidrug resistance in this species is particularly worrisome, especially since the recent description of CTX-M-15 ESBL-producing isolates in Spain. The aim of this study was to characterize a CTX-M-15-producing H. parainfluenzae clinical isolate, HP01, obtained from a urethral swab. METHODS: MICs were determined with gradient strips for this isolate. Hydrolysis assays were performed with the ß LACTA test. Genomic DNA from HP01 was subjected to Illumina and Oxford Nanopore sequencing to investigate the genetic environment of blaCTX-M-15. Phylogenetic analysis was performed with available H. parainfluenzae genomes from the NCBI database, including CTX-M-15 producers. RESULTS: HP01, an XDR isolate, was resistant to penicillin, third-generation cephalosporins, fluoroquinolones, macrolides, cyclines and co-trimoxazole and susceptible only to carbapenems and rifampicin. HP01 carried blaTEM-1, blaCTX-M-15, tet(M), catS and mef(E)/mel and harboured amino acid substitutions in PBP3, PBP5, GyrA, ParC and FolA implicated in resistance. Genomic analysis revealed that blaCTX-M-15 was carried by a Tn3-like transposon inserted into a novel integrative and conjugative element (ICE), ICEHpaSLS, present on the chromosome and belonging to the ICEHin1056 family described in Haemophilus influenzae. The tet(M)-MEGA element was also detected on the chromosome. No plasmid was found. The phylogenetic analysis showed that four H. parainfluenzae producing CTX-M-15 clustered in the same clade. CONCLUSIONS: Here we report the description of an XDR H. parainfluenzae producing blaCTX-M-15 isolated from a urethral swab. The blaCTX-M-15 gene was inserted into an ICE structure similar to those recently described in CTX-M-15 producers in Spain. The emergence of XDR H. parainfluenzae producing blaCTX-M-15 is a matter of great concern. Careful surveillance is required to prevent its spread.

Genome-wide analysis of urogenital and respiratory multidrug-resistant Haemophilus parainfluenzae.

OBJECTIVES: To characterize the mechanisms of antimicrobial resistance and the prevalence of the polysaccharide capsule among urogenital and respiratory Haemophilus parainfluenzae isolates. METHODS: Antimicrobial susceptibility was tested by microdilution. Fifty-five MDR strains were subjected to WGS and were phylogenetically compared with all the available H. parainfluenzae genomes from the NCBI database. The identification of the capsular bexA gene was performed by PCR in 266 non-MDR strains. RESULTS: In 31 of the 42 ampicillin-resistant strains, blaTEM-1 located within Tn3 was identified. ß-Lactamase-negative cefuroxime-resistant strains (n = 12) presented PBP3 substitutions. The catS gene (n = 14), the tet(M)-MEGA element (n = 18) and FolA substitutions (I95L and F154V/S) (n = 41) were associated with resistance to chloramphenicol, tetracycline plus macrolides, and co-trimoxazole, respectively. Thirty-seven isolates had a Tn10 harbouring tet(B)/(C)/(D)/(R) genes with (n = 15) or without (n = 22) catA2. Putative transposons (Tn7076-Tn7079), including aminoglycoside and co-trimoxazole resistance genes, were identified in 10 strains (18.2%). These transposons were integrated into three new integrative and conjugative elements (ICEs), which also included the resistance-associated transposons Tn3 and Tn10. The capsular operon was found only in the urogenital isolates (18/154, 11.7%), but no phylogenetic clustering was observed. The capsular operons identified were similar to those of Haemophilus influenzae serotype c and Haemophilus sputorum type 2. CONCLUSIONS: The identification of ICEs with up to three resistance-associated transposons suggests that these transferable elements play an important role in the acquisition of multidrug resistance in H. parainfluenzae. Moreover, the presence of polysaccharide capsules in some of these urogenital isolates is a cause for concern.

Emergence of multidrug resistance among Haemophilus parainfluenzae from respiratory and urogenital samples in Barcelona, Spain.

Haemophilus parainfluenzae (HPAR) is a Gram-negative bacterium that can become an opportunistic urogenital pathogen. Recently, multidrug resistant (MDR) strains have emerged. We aim to analyse the epidemiology of HPAR at Hospital Universitari de Bellvitge between 2013 and 2017 to determine its putative role in sexually transmitted infections (STI). Strains were classified by sample origin, and antimicrobial susceptibility was performed by disk-diffusion tested on Mueller-Hinton Fastidious. MDR was defined as the resistance of the antimicrobial to three or more antibiotic class. Molecular typing was performed by pulsed-field gel electrophoresis (PFGE) after restriction with SmaI and Cfr9I. We classified 944 HPAR isolates as being of urogenital (n = 175; 18.5%), respiratory (n = 719; 76.2%), or other (n = 50; 5.3%) origins. Among the urogenital isolates, 50 (28.6%) were MDR, which was significantly higher than that found in respiratory samples (40/719; 5.6%; p < 0.01). The frequency of MDR increased progressively among urogenital samples from 13.3% (2013) to 33.3% (2017) (r = 0.8; p = 0.035). The resistance rates for all 944 episodes were significantly higher for cotrimoxazole (51.4%), tetracycline (46.3%), chloramphenicol (28.0%), ciprofloxacin (21.1%), and ampicillin (20.6%). After PFGE, no clonal relationship was found. Clinical charts were available for 40 symptomatic patients with MDR HPAR infections presenting mostly urethritis (n = 26; 65.0%). In all cases, symptoms were treated effectively with combination therapy. Furthermore, in 10 of those patients with urethritis, MDR HPAR was the only potential pathogen to be identified. The emergence of MDR HPAR is a matter of concern, and the detection as a single pathogen highlights its putative role as cause of STI.

Substitutions in PBP3 confer resistance to both ampicillin and extended-spectrum cephalosporins in Haemophilus parainfluenzae as revealed by site-directed mutagenesis and gene recombinants.

Objectives: To determine the association of amino acid substitutions in PBP3 with ß-lactam susceptibility in Haemophilus parainfluenzae. Methods: Single and multiple amino acid mutations at positions 385, 511 and 526 were introduced into PBP3 of a ß-lactam-susceptible H. parainfluenzae strain using site-directed mutagenesis. Recombinants were also generated using PCR-amplified ftsI from clinical strains encoding multiple amino acid substitutions. MICs of ampicillin, cefuroxime, cefotaxime and ceftriaxone were determined using Etest®. Results: Transformation of a susceptible strain with ftsI from clinical strains encoding four substitutions in the transpeptidase region of PBP3 conferred resistance to ampicillin, but not to cephalosporins. Introduction of ftsI from a clinical strain encoding eight substitutions conferred resistance to ampicillin, cefotaxime and ceftriaxone. MICs for recombinants were lower than those for the donor strains. Using site-directed mutagenesis, no single substitution conferred resistance to the tested ß-lactams, although V511A increased the MIC of cefuroxime to the intermediate category for intravenous administration. Recombinants encoding N526K/H/S in combination with V511A were resistant to ampicillin. Substitution S385T increased the MICs of third-generation cephalosporins if V511A was also present. Conclusions: Substitutions in PBP3 are sufficient to confer resistance to both ampicillin and third-generation cephalosporins in H. parainfluenzae. A combination of substitutions at positions Val-511 and Asn-526 confers resistance to ampicillin. Resistance to third-generation cephalosporins probably requires more than four substitutions in PBP3.

Sequencing of 16S rRNA reveals a distinct salivary microbiome signature in Behçet's disease.

Behçet's disease (BD) is characterized by recurrent oro-genital ulcers, mucocutaneous lesions, and serious organ involvement. We investigated the salivary microbiome in BD using high-throughput sequencing of the 16S rRNA V4 region. Stimulated saliva samples were collected from 31 BD patients and 15 healthy controls, and in 9 BD patients, a second saliva sample was collected following dental and periodontal treatment. Sequence analysis identified a total of 908 operational taxonomic units (OTUs) present across all samples. Patients had a microbial community structure that is significantly less diverse than healthy controls. The most overabundant species in BD was Haemophilus parainfluenzae, while the most depleted included Alloprevotella rava and species in the genus Leptotrichia. Periodontal treatment improved oral health indices in BD but had no short-term effect on bacterial community structure. Neither the BD-associated genetic risk locus within the HLA-B/MICA region nor being on immunosuppressive medications explained the differences between patients and controls.

TEM-1-encoding small plasmids impose dissimilar fitness costs on Haemophilus influenzae and Haemophilus parainfluenzae.

Only two beta-lactamases, TEM-1 and ROB-1, have been observed in Haemophilus influenzae, while four different TEM but no ROB enzymes have been found in Haemophilus parainfluenzae. In order to investigate the mechanisms behind the dissemination of small beta-lactamase-encoding plasmids in H. influenzae and H. parainfluenzae, we assessed the fitness cost of three TEM-1- (pPN223, pA1209, pA1606), one TEM-15- (pSF3) and one ROB-1-bearing (pB1000) plasmid when expressed in either bacterial species. All plasmids were stable in H. influenzae and H. parainfluenzae except pB1000, which showed on average (sample mean) 76% curing in H. parainfluenzae after 5  days of subculture. Competition assays between isogenic strains with and without plasmid showed no competitive disadvantage of pPN223 and pA1606 in H. influenzae, or of pA1209 in H. parainfluenzae. In contrast, pSF3 and pB1000 were associated with significant competitive disadvantages in both species. Some of the competitive disadvantages may be related to differences in plasmid copy number and mRNA expression of the beta-lactamase genes, as revealed by quantitative PCR analysis. In conclusion, plasmids encoding TEM beta-lactamases isolated from H. influenzae and H. parainfluenzae can be stably transferred between species. The fast curing of pB1000 in H. parainfluenzae observed in this study correlates to the fact that ROB-1 has never been reported for this species. TEM-1-encoding plasmids are associated with the lowest level of fitness cost, but different TEM-1 plasmids confer different levels of fitness cost on the two hosts.

Emergence of extensively drug-resistant Haemophilus parainfluenzae in Switzerland.

Two homosexual men were colonized in the urethra with Haemophilus parainfluenzae nonsusceptible to ampicillin (MIC, 8 µg/ml), amoxicillin-clavulanate (MIC, 4 µg/ml), cefotaxime (MIC, 1.5 µg/ml), cefepime (MIC, 3 µg/ml), meropenem (MIC, 0.5 µg/ml), cefuroxime, azithromycin, ciprofloxacin, tetracycline, and chloramphenicol (all MICs, ≥ 32 µg/ml). Repetitive extragenic palindromic PCR (rep-PCR) showed that the strains were indistinguishable. The isolates had amino acid substitutions in PBP3, L4, GyrA, and ParC and possessed Mef(A), Tet(M), and CatS resistance mechanisms. This is the first report of extensively drug-resistant (XDR) H. parainfluenzae.

Haemophilus parainfluenzae expresses diverse lipopolysaccharide O-antigens using ABC transporter and Wzy polymerase-dependent mechanisms.

Lipopolysaccharide O-antigens are the basis of serotyping schemes for Gram negative bacteria and help to determine the nature of host-bacterial interactions. Haemophilus parainfluenzae is a normal commensal of humans but is also an occasional pathogen. The prevalence, diversity and biosynthesis of O-antigens were investigated in this species for the first time. 18/18 commensal H. parainfluenzae isolates contain a O-antigen biosynthesis gene cluster flanked by glnA and pepB, the same position as the hmg locus for tetrasaccharide biosynthesis in Haemophilus influenzae. The O-antigen loci show diverse restriction digest patterns but fall into two main groups: (1) those encoding enzymes for the synthesis and transfer of FucNAc4N in addition to the Wzy-dependent mechanism of O-antigen synthesis and transport and (2) those encoding galactofuranose synthesis/transfer enzymes and an ABC transporter. The other glycosyltransferase genes differ between isolates. Three H. parainfluenzae isolates fell outside these groups and are predicted to synthesise O-antigens containing ribitol phosphate or deoxytalose. Isolates using the ABC transporter system encode a putative O-antigen ligase, required for the synthesis of O-antigen-containing LPS glycoforms, at a separate genomic location. The presence of an O-antigen contributes significantly to H. parainfluenzae resistance to the killing effect of human serum in vitro. The discovery of O-antigens in H. parainfluenzae is striking, as its close relative H. influenzae lacks this cell surface component.

Haemophilus parainfluenzae has a limited core lipopolysaccharide repertoire with no phase variation.

Cell surface lipopolysaccharide (LPS) is a well characterized virulence determinant for the human pathogen Haemophilus influenzae, so an investigation of LPS in the less pathogenic Haemophilus parainfluenzae could yield important insights. Using a panel of 18 commensal H. parainfluenzae isolates we demonstrate that the set of genes for inner core LPS biosynthesis largely resembles that of H. influenzae, with an additional heptosyltransferase I gene similar to waaC from Pasteurella multocida. Inner core LPS structure is therefore likely to be largely conserved across the two Haemophilus species. Outer core LPS biosynthetic genes are much less prevalent in H. parainfluenzae, although homologues of the H. influenzae LPS genes lpsB, non-phase variable lic2A and lgtC, and losA1, losB1 and lic2C are found in certain isolates. Immunoblotting using antibodies directed against selected LPS epitopes was consistent with these data. We found no evidence for tetranucleotide repeat-mediated phase variation in H. parainfluenzae. Phosphocholine, a phase variable H. influenzae LPS epitope that has been implicated in disease, was absent in H. parainfluenzae LPS as were the respective (lic1) biosynthetic genes. The introduction of the lic1 genes into H. parainfluenzae led to the phase variable incorporation of phosphocholine into its LPS. Differences in LPS structure between Haemophilus species could affect interactions at the bacterial-host interface and therefore the pathogenic potential of these bacteria.

Detection of blaCTX-M-15 in an integrative and conjugative element in four extensively drug-resistant Haemophilus parainfluenzae strains causing urethritis.

Haemophilus parainfluenzae is a commensal organism with rising numbers of multidrug-resistant (MDR) strains. This pathogen is of increasing clinical relevance in urogenital infection. The aim of this work was to identify and characterise the molecular mechanisms of resistance associated with four cephalosporin-resistant H. parainfluenzae strains collected from patients with urethritis. Antimicrobial resistance was determined by microdilution following European Committee on Antimicrobial Susceptibility Testing criteria. Strains were then analysed by whole-genome sequencing to determine clonal relationship and the molecular basis of antimicrobial resistance. Finally, a phylogenetic analysis was performed on all urogenital MDR strains of H. parainfluenzae previously isolated in our hospital. All strains were resistant to ß-lactams, macrolides, tetracycline, fluoroquinolones, chloramphenicol, cotrimoxazole, and aminoglycosides. The resistance profile was compatible with the presence of an extended-spectrum ß-lactamase (ESBL). Whole-genome sequencing detected blaCTX-M-15 that conferred high minimum inhibitory concentrations to cephalosporins in two novel integrative and conjugative elements (ICEHpaHUB6 and ICEHpaHUB7) that also harboured a blaTEM-1 ß-lactamase. This study shows a novel blaCTX-M-15 ESBL carried in an integrative conjugative element in four extensively drug-resistant H. parainfluenzae strains. This resistance determinant could be transmitted to other sexually transmitted pathogens and this is a cause for concern.

Gastrointestinal microbiome signatures of pediatric patients with irritable bowel syndrome.

BACKGROUND & AIMS: The intestinal microbiomes of healthy children and pediatric patients with irritable bowel syndrome (IBS) are not well defined. Studies in adults have indicated that the gastrointestinal microbiota could be involved in IBS. METHODS: We analyzed 71 samples from 22 children with IBS (pediatric Rome III criteria) and 22 healthy children, ages 7-12 years, by 16S ribosomal RNA gene sequencing, with an average of 54,287 reads/stool sample (average 454 read length = 503 bases). Data were analyzed using phylogenetic-based clustering (Unifrac), or an operational taxonomic unit (OTU) approach using a supervised machine learning tool (randomForest). Most samples were also hybridized to a microarray that can detect 8741 bacterial taxa (16S rRNA PhyloChip). RESULTS: Microbiomes associated with pediatric IBS were characterized by a significantly greater percentage of the class γ-proteobacteria (0.07% vs 0.89% of total bacteria, respectively; P < .05); 1 prominent component of this group was Haemophilus parainfluenzae. Differences highlighted by 454 sequencing were confirmed by high-resolution PhyloChip analysis. Using supervised learning techniques, we were able to classify different subtypes of IBS with a success rate of 98.5%, using limited sets of discriminant bacterial species. A novel Ruminococcus-like microbe was associated with IBS, indicating the potential utility of microbe discovery for gastrointestinal disorders. A greater frequency of pain correlated with an increased abundance of several bacterial taxa from the genus Alistipes. CONCLUSIONS: Using 16S metagenomics by PhyloChip DNA hybridization and deep 454 pyrosequencing, we associated specific microbiome signatures with pediatric IBS. These findings indicate the important association between gastrointestinal microbes and IBS in children; these approaches might be used in diagnosis of functional bowel disorders in pediatric patients.

Rapid detection of Haemophilus influenzae and Haemophilus parainfluenzae in nasopharyngeal swabs by multiplex PCR.

OBJECTIVE: To establish multiplex PCR-based assays for detecting H.influenzae and H.parainfluenzae. And the PCR-based assays were applied to detect the carriage rates of H.influenzae and H.parainfluenzae in nasopharyngeal swab specimens which were collected from healthy children. METHODS: Multiplex primers for species-specific PCR were designed by using DNAstar soft based on the sequences of 16S rRNA genes from genus Haemophilus to detect H.influenzae and H.parainfluenzae. RESULTS: The sensitivity of the 16S rRNA PCR assay for detecting H.influenzae and H.parainfluenzae was 97.53% and 100% respectively, and the specificity was 95.89% and 96.63% respectively. Youden's Index on the ability to detect H.influenzae and H.parainfluenzae was 0.9342 and 0.9663 respectively. 666 nasopharyngeal swab specimens were collected from healthy children. The detection rates of H.influenzae and H.parainfluenzae were 14.11% and 16.07% respectively by using isolation and culture methods. The detection rates of H.influenzae and H.parainfluenzae were 43.54% and 57.96% respectively by 16S rRNA PCR assays. The carriage rates of serotypes a, b, c, d, e, f and non-typeable isolates were 0% (0/666), 0.15% (1/666), 1.20% (8/666), 0.15% (1/666), 1.20% (8/666), 1.80% (12/666), 95.50% (636/666) respectively. CONCLUSION: The multiplex PCR assays were very rapid, reliable and feasible methods for detection of H.influenzae and H.parainfluenzae in pharyngeal swab specimens which were compared to conventional isolation and culture methods. 95.5% of H.influenzae strains in healthy children were nontypeable. The encapsulated or typable strains were mainly three serotypes which was c, e, and f serotype.

Crystal structure of the anti-CRISPR, AcrIIC4.

Clustered regularly interspaced short palindromic repeats (CRISPRs)-CRISPR-associated protein systems are bacterial and archaeal defense mechanisms against invading elements such as phages and viruses. To overcome these defense systems, phages and viruses have developed inhibitors called anti-CRISPRs (Acrs) that are capable of inhibiting the host CRISPR-Cas system via different mechanisms. Although the inhibitory mechanisms of AcrIIC1, AcrIIC2, and AcrIIC3 have been revealed, the inhibitory mechanisms of AcrIIC4 and AcrIIC5 have not been fully understood and structural data are unavailable. In this study, we elucidated the crystal structure of Type IIC anti-CRISPR protein, AcrIIC4. Our structural analysis revealed that AcrIIC4 exhibited a helical bundle fold comprising four helixes. Further biochemical and biophysical analyses showed that AcrIIC4 formed a monomer in solution, and monomeric AcrIIC4 directly interacted with Cas9 and Cas9/sgRNA complex. Discovery of the structure of AcrIIC4 and their interaction mode on Cas9 will help us elucidate the diversity in the inhibitory mechanisms of the Acr protein family.

Metapangenomics of the oral microbiome provides insights into habitat adaptation and cultivar diversity.

BACKGROUND: The increasing availability of microbial genomes and environmental shotgun metagenomes provides unprecedented access to the genomic differences within related bacteria. The human oral microbiome with its diverse habitats and abundant, relatively well-characterized microbial inhabitants presents an opportunity to investigate bacterial population structures at an ecosystem scale. RESULTS: Here, we employ a metapangenomic approach that combines public genomes with Human Microbiome Project (HMP) metagenomes to study the diversity of microbial residents of three oral habitats: tongue dorsum, buccal mucosa, and supragingival plaque. For two exemplar taxa, Haemophilus parainfluenzae and the genus Rothia, metapangenomes reveal distinct genomic groups based on shared genome content. H. parainfluenzae genomes separate into three distinct subgroups with differential abundance between oral habitats. Functional enrichment analyses identify an operon encoding oxaloacetate decarboxylase as diagnostic for the tongue-abundant subgroup. For the genus Rothia, grouping by shared genome content recapitulates species-level taxonomy and habitat preferences. However, while most R. mucilaginosa are restricted to the tongue as expected, two genomes represent a cryptic population of R. mucilaginosa in many buccal mucosa samples. For both H. parainfluenzae and the genus Rothia, we identify not only limitations in the ability of cultivated organisms to represent populations in their native environment, but also specifically which cultivar gene sequences are absent or ubiquitous. CONCLUSIONS: Our findings provide insights into population structure and biogeography in the mouth and form specific hypotheses about habitat adaptation. These results illustrate the power of combining metagenomes and pangenomes to investigate the ecology and evolution of bacteria across analytical scales.

Assessment of trimethoprim-sulfamethoxazole susceptibility testing methods for fastidious Haemophilus spp.

OBJECTIVES: To compare the determinants of trimethoprim-sulfamethoxazole resistance with established susceptibility values for fastidious Haemophilus spp., to provide recommendations for optimal trimethoprim-sulfamethoxazole measurement. METHODS: We collected 50 strains each of Haemophilus influenzae and Haemophilus parainfluenzae at Bellvitge University Hospital. Trimethoprim-sulfamethoxazole susceptibility was tested by microdilution, E-test and disc diffusion using both Mueller-Hinton fastidious (MH-F) medium and Haemophilus test medium (HTM) following EUCAST and CLSI criteria, respectively. Mutations in folA, folP and additional determinants of resistance were identified in whole-genome-sequenced isolates. RESULTS: Strains presented generally higher rates of trimethoprim-sulfamethoxazole resistance when grown on HTM than on MH-F, independent of the methodology used (average MIC 2.6-fold higher in H. influenzae and 1.2-fold higher in H. parainfluenzae). The main resistance-related determinants were as follows: I95L and F154S/V in folA; 3- and 15-bp insertions and substitutions in folP; acquisition of sul genes; and FolA overproduction potentially linked to mutations in -35 and -10 promoter motifs. Of note, 2 of 19 H. influenzae strains (10.5%) and 9 of 33 H. parainfluenzae strains (27.3%) with mutations and assigned as resistant by microdilution were inaccurately considered susceptible by disc diffusion. This misinterpretation was resolved by raising the clinical resistance breakpoint of the EUCAST guidelines to ≤30 mm. CONCLUSIONS: Given the routine use of disc diffusion, a significant number of strains could potentially be miscategorized as susceptible to trimethoprim-sulfamethoxazole despite having resistance-related mutations. A simple modification to the current clinical resistance breakpoint given by the EUCAST guideline for MH-F ensures correct interpretation and correlation with the reference standard method of microdilution.

Prevalence of susceptibility patterns of opportunistic bacteria in line with CLSI or EUCAST among Haemophilus parainfluenzae isolated from respiratory microbiota.

The application of CLSI and EUCAST guidelines led to many discrepancies. Various doubts have already appeared in preliminary stages of microbiological diagnostics of Haemophilus spp. A total of 87 H. parainfluenzae isolates were obtained from throat or nasopharyngeal swabs from adults 18 to 70 years old, both healthy volunteers and patients with chronic diseases between 2013 to 2015 in eastern Poland. Haemophilus spp. were identified by colony morphology, Gram-staining, API NH and MALDI-TOF MS technique. Both susceptibility to various antimicrobials and phenotypes of Haemophilus spp. resistance to beta-lactams were determined. Statistically significant association between applied guidelines and drug resistance patterns were observed to as follows: ampicillin, cefuroxime, cefotaxime, amoxicillin-clavulanate, azithromycin, tetracycline and trimethoprim-sulfamethoxazole. Resistance phenotypes according to CLSI vs. EUCAST were as follows: 3.4% vs. 8.0% for BLNAR and 6.9% vs. 19.5% for BLPACR isolates. In conclusion, this is the first study that reports comparative analysis of drug susceptibility interpretation using CLSI and EUCAST of haemophili rods from human respiratory microbiota in Poland. In case of susceptible, increased exposure (formerly intermediate) category of susceptibility within H. parainfluenzae isolates we have observed EUCAST as more restrictive than CLSI. Moreover, BLNAI and BLPAI phenotype isolates have been observed, as well as BLPBR using only CLSI or EUCAST guidelines, respectively.

Identification of polysaccharide capsules among extensively drug-resistant genitourinary Haemophilus parainfluenzae isolates.

The human commensal Haemophilus parainfluenzae is emerging as an opportunistic multidrug-resistant pathogen. The objectives of this work were to characterise a new capsular operon of extensively drug-resistant (XDR) H. parainfluenzae clinical isolates and study their resistance mechanisms using whole-genome sequencing. All strains were resistant to: ß-lactams, via amino acid changes in PBP3 (S385T, I442F, V511A, N526K and V562I); quinolones, by alterations in GyrA (S84F and D88Y) and ParC (S84F and S138T); chloramphenicol, through the presence of catS; macrolides, via the presence of mel and mef(E)-carrying MEGA element; and tetracycline, through the presence of tet(M) and/or tet(B). Phylogenetic analysis revealed high genomic diversity when compared to the H. parainfluenzae genomes available on the NCBI, the isolates from this study being closely related to the Swiss XDR AE-2096513. A full capsular operon showing homology to that of H. influenzae was identified, in accordance with the observation of a capsular structure by TEM. This study describes for the first time a capsular operon in H. parainfluenzae, a major determinant of pathogenicity that may contribute to increased virulence in XDR clinical isolates. Moreover, phylogenetic analysis suggests the possible spread of an XDR-encapsulated strain in Europe.

Characterization of extended-spectrum beta-lactamase-producing isolates of Haemophilus parainfluenzae.

OBJECTIVES: To characterize the beta-lactam resistance mechanisms of two clinical isolates of cefotaxime-resistant Haemophilus parainfluenzae recovered from patients in South Africa. METHODS: The relatedness of isolates and plasmids was assessed using PFGE and restriction enzyme analysis, respectively. Plasmid-mediated and chromosomally integrated bla(TEM) genes and ftsI genes were sequenced, and the plasmid-mediated bla(TEM-15) was used to transform a range of control organisms. RESULTS: The two isolates were found to be unique according to PFGE, but had an identical 3.7 kb plasmid encoding a TEM-15 beta-lactamase. Both isolates also had substitutions in penicillin binding protein 3 (PBP3) consistent with substitutions known to exist in beta-lactamase-negative ampicillin-resistant (BLNAR) strains of Haemophilus influenzae. The cefotaxime MICs for control strains of H. influenzae, H. parainfluenzae and BLNAR H. influenzae transformed with the plasmid-mediated bla(TEM-15) were 1.0, 1.0 and 4.0 mg/L, respectively, compared with 16.0 and 8.0 mg/L, respectively, for the two parent H. parainfluenzae. CONCLUSIONS: The high-level cefotaxime resistance in the H. parainfluenzae isolates was due to a combination of a plasmid-mediated TEM-15 extended-spectrum beta-lactamase with altered PBP3 probably contributing. Other contributing resistance mechanisms could not be excluded.

Diversity of antibiotic resistance integrative and conjugative elements among haemophili.

The objective of this study was to investigate the sequence diversity in a single country of a family of integrative and conjugative elements (ICEs) that are vectors of antibiotic resistance in Haemophilus influenzae and Haemophilus parainfluenzae, and test the hypothesis that they emerged from a single lineage. Sixty subjects aged 9 months - 13 years were recruited and oropharyngeal samples cultured. Up to 10 morphologically distinct Pasteurellaceae spp. were purified, and then the species were determined and differentiated by partial sequence analysis of 16S rDNA and mdh, respectively. ICEs were detected by PCR directed at five genes distributed evenly across the ICE. These amplicons were sequenced and aligned by the neighbour-joining algorithm. A total of 339 distinguishable isolates were cultured. ICEs with all 5 genes present were found in 9 of 110 (8 %) H. influenzae and 21 of 211 (10 %) H. parainfluenzae, respectively. ICEs were not detected among the other Pasteurellaceae. A total of 20 of 60 (33 %) children carried at least 1 oropharyngeal isolate with an ICE possessing all 5 genes. One of the five genes, integrase, however, consisted of two lineages, one of which was highly associated with H. influenzae. The topology of neighbour-joining trees of the remaining four ICE genes was compared and showed a lack of congruence; though, the genes form a common pool among H. influenzae and H. parainfluenzae. This family of antibiotic resistance ICEs was prevalent among the children studied, was genetically diverse, formed a large gene pool, transferred between H. influenzae and H. parainfluenzae, lacked population structure and possessed features suggestive of panmixia, all indicating it has not recently emerged from a single source.