Antibiotic Susceptibility and Molecular Typing of Invasive Haemophilus influenzae Isolates, with Emergence of Ciprofloxacin Resistance, 2017-2021, Italy.

Haemophilus influenzae invasive disease is a severe infection that needs rapid antibiotic therapy. The aim of the study was to perform and evaluate the serotype distribution, antibiotic susceptibility and molecular characteristics of 392 H. influenzae invasive isolates collected during 2017-2021 in Italy. The majority of isolates were NTHi (305/392, 77.8%), followed by Hib (49/392, 12.5%). Ampicillin resistance was frequently detected (85/392, 21.7%): 12.2% were ß-lactamase producers (all blaTEM except one blaROB), 9.4% were ß-lactamase-negative ampicillin-resistant (BLNAR), with mutations in the ftsI gene. Six isolates were resistant to ciprofloxacin, with substitutions in GyrA and ParC. An MLST analysis revealed the occurrence of international resistant clones, such as ST103 and ST14, highlighting the importance of molecular surveillance.

Nasopharyngeal carriage of Haemophilus influenzae among adults with co-morbidities.

After the widespread use of Haemophilus influenzae type b (Hib) vaccine, H. influenzae invasive disease is now commonly due to non-encapsulated (NTHi), affecting mostly the youngest and the elderly. The objective of this study was to investigate H. influenzae nasopharyngeal carriage rate in adults with co-morbidities and possible associated risk factors. METHODS: Patients aged >50 years with co-morbidities attending medical centres were examined. A nasopharyngeal swab was analysed for H. influenzae presence by cultural and molecular methods (RT-PCR). Univariable and multivariable analysis of risk factors for H. influenzae carriage were performed. Serotype of isolates was determined by PCR capsular genotyping. Minimum inhibitory concentration (MIC) was determined by MIC gradient test and ß-lactamase production was detected by the nitrocephin test. Genotyping was performed by Multilocus sequence typing (MLST). Phylogenetic relationships among carriage and invasive NTHi strains were assessed. RESULTS: Among 248 enrolled patients (median age: 73 years), the carriage rate was 5.6% and 10.5% by cultural method or RT-PCR, respectively. Colonization with H. influenzae was significantly associated with the presence of acute respiratory symptoms (adjusted OR = 12.16, 95% CI: 3.05-48.58, p < 0.001). All colonizing isolates were NTHi. Three isolates (3/14, 21.4%) were resistant to ampicillin and beta-lactamase positive. MLST revealed a high degree of genetic diversity, with 11 different STs from 14 isolates. Eight out of the 11 (72.7%) STs were shared among carriage and invasive isolates. CONCLUSIONS: Adults ≥50 years old with co-morbidities are occasionally colonized by H. influenzae, even if the presence of co-morbidities is not a risk factor for colonization. The presence of acute respiratory symptoms is the only factor associated with H. influenzae colonization. Colonizing H. influenzae are all NTHi. Colonizing H. influenzae often belong to the same STs of invasive disease isolates.

Invasive Haemophilus influenzae type b (Hib) disease in children in Italy, after 20 years of routine use of conjugate Hib vaccines.

Haemophilus influenzae serotype b (Hib) was the leading cause of bacterial meningitis in children before the implementation of infant immunization with conjugate Hib vaccines. Despite the effectiveness of the vaccine, invasive Hib disease cases (i.e. isolation of Hib from a normally sterile site) are still reported in children. All invasive Hib disease cases in children ≤ 15 years reported through the National Surveillance System of Invasive Bacterial Disease, during 2012-2018 in Italy, were analyzed. Hib PCR-confirmed isolates were subjected to MLST and PFGE analysis. The number of copies of the capb locus, a virulence factor potentially contributing to true vaccine failures (TVFs), was determined by Southern blot analysis. Vaccine effectiveness (VE) was determined using a multiple Poisson regression model. 31 cases of invasive Hib disease in children were reported. Fourteen children were vaccinated (TVFs), 14 were unvaccinated and 2 partially vaccinated (vaccination status was unknown for 1 case). The median age of children was 12 months (range 3 months-15 years). A decrease in vaccination coverage was observed in 2014-2016 (source Ministry of Health), and a rise in incidence was documented from 2016 until 2018, especially in children < 5 years. Vaccine effectiveness was estimated to be 83% (95% CI:45-95). 24 isolates were available. The predominant ST was ST6 (70.8%). Cluster analysis of ST6 isolates by PFGE identified five variants. Six isolates (25%) contained multiple copies of the capb locus distributed among TVFs (30%) and unvaccinated children (16.7%). Our data show that both failures to vaccinate and TVFs are associated with invasive Hib disease in children in Italy, during the vaccination era. Most cases in children ≤ 2 years were vaccine-preventable, since they occurred in unvaccinated subjects (13/21 cases, 62%). No host predisposing factors for TVF were recognized. TVFs were not significantly associated with either specific genotypes or amplification status of the capb locus.

Increasing trend in invasive non-typeable Haemophilus influenzae disease and molecular characterization of the isolates, Italy, 2012-2016.

Routine immunization of infants with conjugate vaccines against Haemophilus influenzae type b (Hib) has greatly reduced the incidence of invasive Hib disease; however changes in the epidemiology of H. influenzae disease have occurred. We describe the epidemiology of invasive H. influenzae disease and the characterization of isolates collected in Italy between 2012 and 2016. Trends in the overall incidence of invasive H. influenzae disease were calculated. Isolates were characterized by PCR capsular genotyping, antimicrobial susceptibility testing, ampicillin resistance-associated gene sequencing and MLST. Trends in incidence by serotype and serotype-specific distribution were estimated using multiple imputation of missing data. The overall incidence of invasive H. influenzae disease increased 22.5% yearly (from 0.11/100,000 in 2012 to 0.24/100,000 in 2016). Most cases (82.0%) were due to non-typeable H. influenzae (NTHi). An increasing trend in NTHi disease burden was estimated; the highest rise was among infants <12 months (40.8% annual increase). Invasive Hib disease showed a fluctuating trend with a clear increase in 2016, while we found an increasing trend for disease due to non-Hib capsulated serotypes in the elderly (32.9% annual increase). Ampicillin resistance mediated by either ß-lactamase or altered penicillin-binding proteins 3 (PBP3) increased. In spite of genetic diversity of NTHi, sequence types (STs) associated with ampicillin resistance status were identified (ST103/ST106 linked to ß-lactamase production and ST14 linked to a specific PBP3 substitution pattern). The increasing trend in invasive NTHi disease in infants is of concern underlying the need for the development of a future vaccine against NTHi.

Emergence of Invasive Haemophilus influenzae Type A Disease in Italy.

We report on the first detection of 2 cases of invasive Haemophilus influenzae type a (Hia) disease in Italy. The cases were sustained by the same Hia "strain" belonging to the ST23 clone that has previously been reported only outside Europe. The emergence of invasive Hia disease is of concern.

First Whole-Genome Sequence of a Haemophilus influenzae Type e Strain Isolated from a Patient with Invasive Disease in Italy.

In the present era of conjugate vaccines against Haemophilus influenzae type b, non-vaccine-preventable strains are of concern. Here, we report the first whole-genome sequence of an invasive H. influenzae type e strain. This genomic information will enable further investigations on encapsulated non-type b H. influenzae strains.

Carriage of Haemophilus influenzae in the oropharynx of young children and molecular epidemiology of the isolates after fifteen years of H. influenzae type b vaccination in Italy.

BACKGROUND: Haemophilus influenzae is an important pathogen able to cause a wide spectrum of diseases in children. Colonization of the upper respiratory tract is a risk factor for developing disease. This study aimed to investigate the oropharyngeal carriage rate of H. influenzae in young children in two Italian cities, 15 years after H. influenzae type b (Hib) vaccination was introduced. Antibiotic resistant traits and genotypes of the colonizing H. influenzae isolates were investigated. METHODS: Oropharyngeal swabs were obtained from 717 healthy children aged <6 years (June 2012-July 2013). Potential risk factors for H. influenzae colonization were investigated. H. influenzae isolates from carriage were characterized by PCR capsular typing, ampicillin susceptibility testing, resistance-associated gene sequencing and multilocus sequence typing (MLST). For comparison purposes, 38 non-typeable H. influenzae (NTHi) isolates from invasive disease were genotyped by MLST. RESULTS: The overall H. influenzae carriage rate was 14.1% (101/717). Age, study site, presence of young siblings, and complete Hib vaccination status were independently associated with colonization. Of 101 isolates, 98 were NTHi, 2 were type e and 1 was type f. The overall ampicillin resistance rate was 15.8% (16/101). Resistance was mediated by TEM-1 ß-lactamase production in half of isolates (n=8) or modifications in penicillin-binding protein (PBP) 3 in the other half (n=8). Several substitutions were discovered in PBP3 including the Asn526Lys change. Seventy-six different STs were identified among 98 NTHi isolates from carriage, with only 4 STs (ST12, ST57, ST238, ST1238) encompassing ≥ 3 isolates. Comparison of carriage and disease isolates found that several STs were shared between the two sources, although none of the major disease-associated STs were observed in carriage isolates. CONCLUSIONS: NTHi is the predominant serotype in carriage. The importance of monitoring both NTHi colonization rate and circulating genotypes should be emphasized in the era of the Hib conjugate vaccines.

First report of neonatal bacteremia caused by "Haemophilus quentini" diagnosed by 16S rRNA gene sequencing, Italy.

We report the first case of neonatal bacteremia caused by a "Haemophilus quentini" isolate in Italy. The isolate was differentiated from H. influenzae by 16S rRNA sequencing and was characterized by comparison with the wild-type "H. quentini" CCUG 36167. Both isolates carried substitutions in penicillin-binding protein 3 but were susceptible to aminopenicillins.

Genital carriage of the genus Haemophilus in pregnancy: species distribution and antibiotic susceptibility.

Recent reports have hypothesized that colonization of the maternal genital tract with non-capsulated Haemophilus influenzae could result in neonatal invasive disease. In this study, genital carriage of the genus Haemophilus was investigated in 510 pregnant women attending an Italian hospital for routine controls. Overall, vaginal carriage of the genus Haemophilus was 9.0 % (46/510). A high colonization rate with Haemophilus parainfluenzae (37/510, 7.3 %) was found; other species, such as Haemophilus pittmaniae (7/510, 1.4 %) and Haemophilus haemolyticus (2/510, 0.4 %), were detected for the first time in the genital flora by 16S rRNA gene sequencing. Notably, no H. influenzae was identified, in agreement with previous investigations indicating that this species is rarely isolated from the genito-urinary tract of pregnant women. No antibiotic resistance was detected in H. pittmaniae and H. haemolyticus, but quite a high degree of ampicillin (10/37, 27 %) and ciprofloxacin (3/37, 8.1 %) resistance was observed in H. parainfluenzae. Five ampicillin-resistant isolates were ß-lactamase producers, whereas five isolates exhibited a ß-lactamase-negative ampicillin-resistant (BLNAR) phenotype. Sequencing of penicillin-binding protein 3 revealed that Val511Ala, Asn526Ser, Ala530Ser and Thr574Ala changes were associated with BLNAR phenotypes. Two ciprofloxacin-resistant isolates carried substitutions in both GyrA (Ser84Phe and Asp88Tyr) and ParC (Ser84Tyr and Met198Leu); the other ciprofloxacin-resistant isolate had substitutions in ParC, only (Ser138Thr and Met198Leu). In conclusion, ∼10 % of pregnant women carried a species of Haemophilus in their genital tract. The emergence of non-ß-lactamase-mediated resistance in genital H. parainfluenzae is a matter of concern because of the risk of mother-to-baby transmission.

Whole-Genome Sequences of Nonencapsulated Haemophilus influenzae Strains Isolated in Italy.

Haemophilus influenzae is an important human pathogen involved in invasive disease. Here, we report the whole-genome sequences of 11 nonencapsulated H. influenzae (ncHi) strains isolated from both invasive disease and healthy carriers in Italy. This genomic information will enrich our understanding of the molecular basis of ncHi pathogenesis.

Identification of Haemophilus influenzae clones associated with invasive disease a decade after introduction of H. influenzae serotype b vaccination in Italy.

The introduction of Haemophilus influenzae serotype b (Hib) conjugate vaccines has changed the epidemiology of invasive H. influenzae disease, with a shift in the predominant serotype from Hib to nonencapsulated H. influenzae (ncHi). The objective of this study was to identify the genotypes/clones associated with invasive H. influenzae disease in Italy. Eighty-seven H. influenzae strains isolated in the years 2009 to 2011 within the National Surveillance of Invasive Bacterial Disease program were analyzed. Strains were characterized by serotyping, antimicrobial susceptibility testing, and multilocus sequence typing (MLST). Genetic polymorphisms in the bla(TEM) gene promoter region as well as the occurrence of both adhesin genes (hmwA and hia) and the IgA1 protease-encoding gene (igaB) were also investigated. Of 87 strains, 67 were ncHi and 20 were encapsulated. Eleven strains were ß-lactamase positive, harboring the bla(TEM) gene. Most bla(TEM) genes (10/11) were associated with a Pdel promoter region exhibiting a 135-bp deletion; the remaining strain possessed the Pa/Pb overlapping promoter. MLST analysis showed that encapsulated isolates were clonal, with each serotype sharing a few related sequence types (STs). Forty-six different STs were identified among the 67 ncHi strains. Despite this heterogeneity, a group of closely related STs (ST103, ST139, and ST145) encompassed almost 25% of all ncHi strains and 45.5% of the ß-lactamase producers carrying the Pdel promoter. These major ST clones were found to be associated with the hmwA gene but not with the igaB gene. To conclude, although the heterogeneity of the ncHi population was confirmed, diffusion of major successful ST clones was documented.

Biofilm-growing intestinal anaerobic bacteria.

Sessile growth of anaerobic bacteria from the human intestinal tract has been poorly investigated, so far. We recently reported data on the close association existing between biliary stent clogging and polymicrobial biofilm development in its lumen. By exploiting the explanted stents as a rich source of anaerobic bacterial strains belonging to the genera Bacteroides, Clostridium, Fusobacterium, Finegoldia, Prevotella, and Veillonella, the present study focused on their ability to adhere, to grow in sessile mode and to form in vitro mono- or dual-species biofilms. Experiments on dual-species biofilm formation were planned on the basis of the anaerobic strains isolated from each clogged biliary stent, by selecting those in which a couple of anaerobic strains belonging to different species contributed to the polymicrobial biofilm development. Then, strains were investigated by field emission scanning electron microscopy and confocal laser scanning microscopy to reveal if they are able to grow as mono- and/or dual-species biofilms. As far as we know, this is the first report on the ability to adhere and form mono/dual-species biofilms exhibited by strains belonging to the species Bacteroides oralis, Clostridium difficile, Clostridium baratii, Clostridium fallax, Clostridium bifermentans, Finegoldia magna, and Fusobacterium necrophorum.

Haemophilus influenzae in children with cystic fibrosis: antimicrobial susceptibility, molecular epidemiology, distribution of adhesins and biofilm formation.

Haemophilus influenzae commonly infects the respiratory tract of patients with cystic fibrosis (CF), early in childhood. In this investigation, 79 H. influenzae isolates were recovered from the respiratory secretions of 64 CF patients (median age: 5 years) included in a 5-year follow-up study. Fifteen of the 64 patients contributed two or more H. influenzae isolates overtime. Serotyping, antibiotic susceptibility testing, genotyping, detection of both hmwA and hia adhesin genes and hypermutable strains was carried out. Biofilm formation ability was investigated. Most strains (72/79, 91.2%) were nonencapsulated or nontypeable (NTHi). Resistance to ampicillin (13.9%) and imipenem (17.7%) was the most detected. Few isolates (2.5%) exhibited the hypermutable phenotype. The NTHi strains showed 55 different genotypes, but 19 clusters of closely related strains were identified. Nine clusters included strains that cross-colonised several patients over a long-time period (mean: 3.7 years). Most patients with sequential isolates harboured strains genetically unrelated, but persistent colonisation with the same clone was observed in 37.5% of patients. Over 45% of NTHi strains contained hmwA-related sequences, 26.3%, hia, 8.3% both hmwA and hia, while 19.4% lacked both. A significant association was found between occurrence of an adhesive gene (irrespective of which) and both persistence (P<0.0001) and long-term cross-colonisation (P<0.0001). Mean biofilm level formed by the persistent strains was found significantly increased compared to non-persistent ones (P<0.0001). Hia-positive strains produced significantly more biofilm than hmwA-carrying strains (P<0.01). Although a high turnover of NTHi strains in FC patients was observed, distinct clones with increased capacity of persistence or cross-colonisation occurred.

Ten years of Hib vaccination in Italy: prevalence of non-encapsulated Haemophilus influenzae among invasive isolates and the possible impact on antibiotic resistance.

The introduction of Haemophilus influenzae type b (Hib) conjugate vaccines has greatly reduced the incidence of invasive Hib disease. However, concern exists about the possible emergence of "strain replacement". We report the epidemiology and characterization of isolates from invasive H. influenzae disease in Italy through 2007-2009, 10 years after Hib vaccination was introduced. Invasive H. influenzae disease cases were detected through the National Surveillance of Invasive Bacterial Disease. Seventy-eight H. influenzae strains were serotyped and tested for antimicrobial susceptibility. Genetic basis of resistance to ß-lactams was investigated. The annual incidence of invasive H. influenzae infection was 0.06/100,000 in 2007, 0.08/100,000 in 2008 and 0.09/100,000 in 2009 in all age groups. A slight increase in disease incidence has been observed in adults ≥65 years since 2007. Nonencapsulated (ncHi) predominated among H. influenzae isolates from all age groups: 61.5%, 76.0%, and 75.0% for <5, 5-64 and ≥65 years, respectively. Although ncHi mainly caused bacteremia, meningitis due to ncHi increased in comparison with previous data (38.6% in 2007-2009 vs. 26.2% 1997-2002). Prevalence of encapsulated non-Hib strains grew significantly (4.1% in 1997-2002 vs.16.7% in 2007-2009; p<0.001), although they remained rare. Resistance to ampicillin mediated by ß-lactamase declined, but that due to altered penicillin-binding protein 3 increased. In conclusion, routine use of Hib vaccines produced both a drastic decrease in the number of invasive H. influenzae cases and epidemiological changes in disease. Overall, pediatric H. influenzae disease has become less common whereas there has been a slight increase of disease in the elderly. A marked change in the predominant serotype from Hib to ncHi has occurred. Changes in the H. influenzae population moderately affected antibiotic resistance trends.

Microbial biofilms associated with biliary stent clogging.

Endoscopic stenting is a palliative approach for the treatment of diseases involving biliary obstruction. Its major limitation is represented by stent occlusion, followed by life-threatening cholangitis, often requiring stent removal and replacement. Although it has been suggested that microbial colonization of biliary stents could play a role in the clogging process, the so far available data, particularly on the role of anaerobic bacteria, are not enough for a comprehensive description of this phenomenon. Our study was focused on the analysis of 28 explanted biliary stents by culturing, denaturing gradient gel electrophoresis and scanning electron microscopy to identify all the aerobic/anaerobic bacteria and fungi involved in the colonization of devices and to verify the ability of isolated anaerobic bacterial strains to form a biofilm in order to better understand the mechanisms of stent clogging.

Genetic characterization of the capsulation locus of Haemophilus influenzae serotype e.

The capsulation (cap) locus of Haemophilus influenzae type e (Hie) was characterized and sequenced. No IS1016 element was found to flank the locus. The 18.2-kb locus included 14 open reading frames (ORFs), which were grouped into three functional regions. Eight new ORFs (named ecs1 to ecs8) were identified in the Hie capsule-specific region II.

Haemophilus parainfluenzae meningitis in an adult associated with acute otitis media.

A case of Haemophilus parainfluenzae meningitis in a woman with a 3-day history of acute otitis media is reported. Her medical history included bladder cancer surgery. Unequivocal identification of the isolate was obtained by using molecular techniques such as 16S rRNA sequencing besides to conventional culture methods. To investigate bacterial virulent traits possibly related to invasive properties, in vitro serum resistance of our isolate was analyzed, but it was found serum susceptible. Our study demonstrates that H. parainfluenzae can be considered an opportunistic pathogen able to cause life-threatening infections not only in children but also in patients with underlying conditions.

Variation in expression of HMW1 and HMW2 adhesins in invasive nontypeable Haemophilus influenzae isolates.

BACKGROUND: Among surface antigens of nontypeable Haemophilus influenzae (NTHi), the HMW1 and HMW2 proteins are the major adhesins promoting colonization of the upper respiratory tract. Since they are potential vaccine candidates, knowledge concerning variation in HMW proteins expression among clinical isolates is of great interest. In this study, expression of hmw1A and hmw2A genes was evaluated by quantitative real-time reverse transcription-PCR in 3 NTHi invasive isolates (strains 56, 72, 91) and in the prototype strain 12. Number of 7-bp repeats within the hmwA promoters and presence of HMW proteins by Western blotting were also determined. RESULTS: Results showed that gene transcription varied not only among different isolates but also between the hmw1A and hmw2A genes from the same isolate. Compared to that found in prototype strain 12, up-regulation of the hmw1A gene expression was found in strain 56, down-regulation of both hmw1A and hmw2A genes transcripts was observed in strain 72 whereas the two hmwA genes appeared differentially expressed in strain 91 with the hmw1A transcript enhanced but the hmw2A transcript reduced. CONCLUSION: Increasing numbers of 7-bp repeats within the hmwA promoters generally correlated with decreased amounts of mRNA transcript, however additional control mechanisms contributing to modulation of hmw1A gene seem to be present.