BLNAR Strains Research Articles

Epidemic Trends and Biofilm Formation Mechanisms of Haemophilus influenzae: Insights into Clinical Implications and Prevention Strategies.

Haemophilus influenzae (H. influenzae) is a significant pathogen responsible for causing respiratory tract infections and invasive diseases, leading to a considerable disease burden. The Haemophilus influenzae type b (Hib) conjugate vaccine has notably decreased the incidence of severe infections caused by Hib strains, and other non-typable H. influenzae (NTHi) serotypes have emerged as epidemic strains worldwide. As a result, the global epidemic trends and antibiotic resistance characteristics of H. influenzae have been altered. Researches on the virulence factors of H. influenzae, particularly the mechanisms underlying biofilm formation, and the development of anti-biofilm strategies hold significant clinical value. This article provides a summary of the epidemic trends, typing methods, virulence factors, biofilm formation mechanisms, and prevention strategies of H. influenzae. The increasing prevalence of NTHi strains and antibiotic resistance among H. influenzae, especially the high β-lactamase positivity and the emergence of BLNAR strains have increased clinical difficulties. Understanding its virulence factors, especially the formation mechanism of biofilm, and formulating effective anti-biofilm strategies may help to reduce the clinical impact. Therefore, future research efforts should focus on developing new approaches to prevent and control H. influenzae infections.

Use of trans-complementation method to determine the effects of various ftsI mutations on β-lactamase-negative ampicillin-resistant (BLNAR) Haemophilus influenzae strains

Haemophilus influenzae is a causative agent of serious infections, especially among children. β-lactam antibiotics are commonly used for the treatment of these infections. Among H. influenzae isolates, β-lactam resistance is due to the presence of β-lactamase, or to mutations in the ftsI gene that generate altered PBP3 (penicillin-binding protein 3) with reduced affinity for β-lactams (BLNAR-β-lactamase-negative, ampicillin-resistant). Wild-type ftsI gene encoding for PBP3 was amplified in whole from β-lactam susceptible H. influenzae Rd and cloned in pLS88 plasmid to obtain pADUTAS17, which was then used to transform known BLNAR strains, susceptible strains, and a strain (CF55) with wild-type ftsI but unexplained reduced β-lactam susceptibility. Ampicillin and cefotaxime MICs (minimum inhibitory concentration) were determined after transformation with pLS88 and pADUTAS17 plasmids. The results showed that antibiotic susceptibilities were not affected by trans-complementation for isolates carrying wild-type ftsI gene. However, trans-complementation for all BLNAR strains showed decreases between -0.957 and 0.5-fold for ampicillin and cefotaxime, confirming the role of the PBP3 substitutions in the BLNAR phenotype of these isolates. The first article showed that trans-complementation might be a useful tool in the investigation of decreased β-lactam susceptibility in H. influenzae.

β-Lactamase-non-producing ampicillin-resistant Haemophilus influenzae is acquiring multidrug resistance

BackgroundHaemophilus influenzae strains with reduced susceptibilities to antimicrobial agents have emerged in Japan. Here, we aimed to investigate H. influenzae non-susceptibility to β-lactams and non-β-lactams. MethodsA total of 260 H. influenzae isolates from patients in 2013–2016 were analysed. Antimicrobial susceptibilities were assessed by determining the minimum inhibitory concentration. Additionally, isolates with reduced susceptibility were analysed by both genetic and statistical methods. Resultsβ-Lactamase-non-producing ampicillin-resistant H. influenzae (BLNAR) strains increased significantly and accounted for more than 50% of all isolates from 2014. Additionally, the proportion of quinolone-low-susceptibility isolates increased significantly (P<0.05). Among these, three quinolone-non-susceptible isolates showed minimum inhibitory concentrations higher than the susceptibility breakpoint of levofloxacin. Moreover, one of the three isolates showing multidrug resistance was resistant to macrolides, β-lactams, and quinolones. Low susceptibilities to non-β-lactams were significantly associated with BLNAR. ConclusionsThe present study indicates that BLNAR strains are increasing and tend to show multidrug resistance. Additionally, multidrug-resistant H. influenzae (MDRHI) has emerged. To prevent the further spread of MDRHI, the proportions of BLNAR strains should be evaluated.

Development of a highly resolved loop-mediated isothermal amplification method to detect the N526K ftsI mutation of β-lactamase-negative ampicillin-resistant Haemophilus influenzae

Rapid and easy detection of sequence polymorphisms, including nucleotide point mutations of bacterial pathogens responsible for amino acid substitutions linked to drug resistance, is essential for the proper use of antimicrobial agents. Here, a detection method using loop-mediated amplification (LAMP) combined with amplification refractory mutation system (ARMS) to accurately distinguish a different single nucleotide in the target sequence was established, named ARMS-SNP LAMP. This procedure is capable of species-specific detection of a nucleotide (1578T) in the ftsI gene on Haemophilus influenzae without amplifying the sequence carrying the point mutations (T1578G/A) in β-lactamase-negative ampicillin resistant (BLNAR) strains. Reactions were performed at 61°C for 45min. Successful target gene amplifications were detected by measuring real-time turbidity using a turbidimeter and visual detection. The assay had a detection limit of 10.0pg of genomic DNA per reaction and showed specificity against 52 types of pathogens, whereas amplifications were completely blocked in even 100.0ng/μL of genomic DNA with point mutations at T1578G and T1578A. The expected ARMS-SNP LAMP products were confirmed through identical melting curves in real-time LAMP procedures. This novel procedure was also used to analyze 57 clinical isolates of H. influenzae. All 25 clinical isolates with the naïve sequence of 1578T gave positive results. In addition, concordant negative results were obtained for 31 of the BLNAR strains with the T1578G mutation and one strain with the T1578A mutation. The ARMS-SNP LAMP method is a simple and rapid method for SNP-genotyping of a clinical isolate as point-of-care testing (POCT) technology. It is suitable for use in both resource-limited situations and well-equipped clinical settings because of its simplicity and convenience.

Ampicillin resistance in Haemophilus influenzae from COPD patients in the UK.

BackgroundHaemophilus influenzae is commonly isolated from the airways of COPD patients. Antibiotic treatment may cause the emergence of resistant H. influenzae strains, particularly ampicillin-resistant strains, including β-lactamase-negative ampicillin resistance (BLNAR) strains. Genetic identification using ftsI sequencing is the optimum method for identifying mutations within BLNAR strains. The prevalence of BLNAR in COPD patients during the stable state has not been reported. We investigated the antibiotic resistance patterns of H. influenzae present in the sputum of stable COPD patients, focusing on ampicillin resistance; the prevalence of enzyme and non-enzyme-mediated ampicillin resistance was determined. A subset of patients was followed up longitudinally to study H. influenzae strain switching and antibiotic sensitivity changes.Patients and methodsSputum sampling was performed in 61 COPD patients, with 42 samples obtained at baseline; H. influenzae was detected by polymerase chain reaction in 28 samples. In all, 45 patients completed the follow-up for 2 years; 24 H. influenzae isolates were obtained.ResultsDisk diffusion showed the highest antibiotic resistance in the penicillin antibiotic group (eg, 67% for ampicillin) and macrolides (eg, 46% for erythromycin), whereas all isolates were susceptible to quinolones. Of the 16 isolates resistant to ampicillin, 9 (56%) were β-lactamase positive. The β-lactamase-negative isolates were further investigated; none of these fulfilled the phenotypic BLNAR classification criteria of ampicillin minimum inhibitory concentration >1 µg/mL, and only one demonstrated an ftsI mutation. Frequent H. influenzae strain switching was confirmed using multilocus sequence typing and was associated with changes in the antibiotic sensitivity pattern.ConclusionWe observed an overidentification of ampicillin resistance by disk diffusion. The majority of ampicillin resistance was due to enzyme production. H. influenzae strain changes during the stable state may be associated with a change in antibiotic sensitivity; this has implications for empirical antibiotic prescribing.

Nationwide survey of the development of drug resistance in the pediatric field in 2007 and 2010: drug sensitivity of Haemophilus influenzae in Japan (second report).

The Drug-Resistant Pathogen Surveillance Group in Pediatric Infectious Disease conducted national surveillance for Haemophilus influenzae in 2007 (phase 3) and 2010 (phase 4), following the previous surveillance conducted from 2000 to 2001 (phase 1) and in 2004 (phase 2). We examined the antimicrobial susceptibility for H. influenzae derived from clinical specimens of pediatric patients collected nationwide from 27 institutions during phases 3 (386 strains) and 4 (484 strains). The frequency of β-lactamase-nonproducing ampicillin (ABPC)-resistant (BLNAR) strains, which rapidly increased from 11.4 % in phase 1 to 43.4 % in phase 2, has gradually decreased from 38.3 % in phase 3 to 37.8 % in phase 4. In contrast, On the other hand, the frequency of β-lactamase-producing strains, which continuously decreased from 8.3 % in phase 1 to 4.4 % in phase 3, has increased to 8.7 % in phase 4. Prevalence of β-lactamase-producing clavulanic acid/amoxicillin-resistant (BLPACR) strains, especially, has increased from 1.6 % in phase 3 to 4.8 % in phase 4. The oral antimicrobial agents with the lowest MIC90 were levofloxacin in both phases, and tosufloxacin in phase 4 (≤0.063 μg/ml), whereas for intravenous use the corresponding agent was tazobactam/piperacillin in both phases (0.125 μg/ml). There was no increase in the MIC90 of most β-lactams between phase 3 and phase 4. In relationship to sex, age, presence of siblings, attendance at a daycare center, siblings’ attendance at a daycare center, and prior administration of antimicrobial agents within 1 month, the frequency of β-lactamase-nonproducing ABPC-intermediately resistant (BLNAI) strains + BLNAR strains was high (P = 0.005) in cases with prior administration of antimicrobial agents in phase 3.

Microbiological characterization of Streptococcus pneumoniae and non-typeable Haemophilus influenzae isolates as primary causes of acute otitis media in Bulgarian children before the introduction of conjugate vaccines

BackgroundPneumococcal and Haemophilus influenzae type b (Hib) vaccines were introduced in our national immunisation program in April 2010. The aims of this retrospective, laboratory-based study were to determine the serotypes and antibiotic resistance of Streptococcus pneumoniae and H. influenzae isolates from middle ear fluid (MEF) collected before the introduction of immunization.MethodsS. pneumoniae (n = 128) and H. influenzae (n = 40) strains isolated from MEF of children with AOM between 1994 and 2011 were studied. MICs were determined by a microdilution assay. Serotyping of S. pneumoniae was done by Quellung method and PCR capsular typing was used for H. influenzae. Macrolide resistance genes were detected by PCR for erythromycin resistant S. pneumoniae (ERSP). DNA sequencing of ftsI gene was performed for ampicillin nonsusceptible H. influenzae.ResultsThe most common serotypes found among children with pneumococcal AOM were 19 F (20.3%), 6B (15.6%), and 19A (10.9%). The potential coverage rates by the PCV7, PCV10 and PCV13 of children aged < 5 years were 63.6%, 66.4% and 85.5%, respectively. Reduced susceptibility to oral penicillin was seen in 68.1%; resistance to erythromycin was 46.9%. We found erm(B) gene in 56.7% of the ERSP, mef(E) gene in 25%; 15% harbored both genes erm(B) + mef(E) and 3.3% had mutations of L4 ribosomal protein. Of the 40 H. influenzae isolates 97.5% were nontypeable. Nonsusceptibility to ampicillin occurred in 25%. Ampicillin resistance groups were: β-lactamase-positive ampicillin resistant (BLPAR) strains (10%), β-lactamase-negative ampicillin resistant (BLNAR) strains (12.5%) and β-lactamase-positive amoxicillin-clavulanate resistant (BLPACR) strains (2.5%). Among BLNAR and BLPACR most of the isolates (5/6) belonged to group II, defined by the Asn526Lys substitution.ConclusionsThe levels of antibiotic resistance among S. pneumoniae and H. influenzae causing severe AOM in children are high in our settings. The existence of multidrug-resistant S. pneumoniae serotype 19A is of particular concern. The rate of BLNAR and BLPACR strains among H. influenzae isolates was 15%.

Serotypes, antimicrobial susceptibility, and beta-lactam resistance mechanisms of clinical Haemophilus influenzae isolates from Bulgaria in a pre-vaccination period

To determine the serotypes, antimicrobial susceptibility, and beta-lactam resistance mechanisms of Haemophilus influenzae strains isolated from invasive and respiratory tract infections (RTIs) prior to the introduction of Haemophilus influenzae type b (Hib) vaccination in Bulgaria. A total of 259 isolates were serotyped by polymerase chain reaction. Susceptibility to antibiotics and beta-lactamase production were determined, and DNA sequencing of the ftsI gene was performed for ampicillin non-susceptible strains. The invasive H. influenzae infections in children were mainly due to serotype b (94.5% in meningitis and 88.9% in other invasive cases). Non-typeable strains (97.4%) were the most frequently found H. influenzae strains in RTIs both in children and adults. Non-susceptibility to ampicillin occurred in 22% of all strains. Ceftriaxone and levofloxacin were the most active agents tested. Ampicillin resistance occurred in 34.4% of invasive strains, and beta-lactamase production was the only mechanism found. Among respiratory tract isolates, ampicillin non-susceptible strains (18%) were classified into the following groups: beta-lactamase-positive, ampicillin-resistant (BLPAR) strains (7.2%); beta-lactamase-negative, ampicillin-non-susceptible (BLNAR) strains (8.2%); and beta- lactamase-positive, amoxicillin-clavulanate-resistant (BLPACR) strains (2.6%). Among 21 BLNAR and BLPACR strains there were 9 different patterns of multiple-amino acid substitutions in penicillin-binding protein 3. Of these, most isolates (81.0%) belonged to group II, defined by the Asn526Lys substitution. Beta-lactamase production was more common among invasive strains than in respiratory isolates. BLNAR and BLPACR H. influenzae were found only among respiratory tract isolates.

High prevalence of nasal carriage of β-lactamase-negative ampicillin-resistantHaemophilus influenzaein healthy children in Korea

This study investigated the carriage of antimicrobial resistant Haemophilus influenzae in 582 healthy children attending kindergarten or elementary school at four intervals over a 9-month period in Seoul, Korea. Diverse colonization patterns and a lower level of long-term persistent carriage by H. influenzae status were evident in this study. Colonizing H. influenzae isolates showed a high rate of resistance to β-lactams including ampicillin (51·9%), cefaclor (52·1%), and amoxicillin/clavulanate (16·3%). Based on the ampicillin resistance mechanism, H. influenzae isolates were categorized as β-lactamase-negative, ampicillin-susceptible (BLNAS) (48·1%), β-lactamase-positive, ampicillin-resistant (BLPAR) (22·6%), β-lactamase-negative, ampicillin-resistant (BLNAR) (22·8%), and β-lactamase-positive, amoxicillin/clavulanate-resistant (BLPACR) strains (6·5%). This study provides the first evidence of a high prevalence (22·8%) of BLNAR strains of H. influenzae nasal carriage in healthy children attending kindergarten or the first 2 years of elementary school in Korea. The high carriage of these resistant strains in overcrowded urban settings may create reservoirs for development of H. influenzae-resistant strains.

In vitro activity of cefditoren and other comparators against Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis causing community-acquired respiratory tract infections in China

The aim of this study was to evaluate the in vitro activity of cefditoren and comparators against Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis causing community-acquired respiratory tract infections (CARTIs). A total of 391 Streptococcus pneumoniae, 266 H. influenzae, and 76 M. catarrhalis were isolated from 10 centers located at 6 cities in China from January 2009 to May 2010. The microdilution method was used to determine minimum inhibitory concentrations (MICs). The pneumococci comprised 189 (48.3%) penicillin susceptible, 129 (33.0%) penicillin intermediate, and 73 (18.7%) penicillin resistant. Moxifloxacin and levofloxacin showed the highest activity (99.2% and 97.7%, respectively) against Streptococcus pneumoniae, followed by parenteral penicillin G (95.7%), cefditoren (83.1%) and amoxicillin–clavulanic acid (79.3%). Among the 266 H. influenzae isolates, 26 (9.8%) were ampicillin-resistant β-lactamase–producing strains and 24 (9.0%) were ampicillin-resistant β-lactamase–nonproducing strains (BLNAR). Most of antimicrobial agents demonstrated good activity (>97% susceptibility) against H. influenzae except ampicillin, cefuroxime, and cefaclor, which showed relatively lower activity (81.2%, 88.7%, and 88%, respectively). Cefditoren showed excellent activity with the lowest MIC50 and MIC90 (≤0.016/0.064 μg/mL) among all tested drugs, which is independent of β-lactamase production or ampicillin resistance. Cefditoren at a concentration of 0.5 μg/mL inhibited all BLNAR strains. Seventy of 76 isolates of M. catarrhalis produced β-lactamase. Cefditoren also showed excellent activity with MIC90 of 0.064 μg/mL against β-lactamase–nonproducing strains and 0.5 μg/mL against β-lactamase–producing strains. In conclusion, the excellent intrinsic activity of cefditoren suggests that it may be a good choice for the treatment of CARTIs caused by Streptococcus pneumoniae, H. influenzae, and M. catarrhalis in China, while the activity should be closely monitored.

An evaluation of SNP-based PCR methods for the detection of β-lactamase-negative ampicillin-resistant Haemophilus influenzae

Forty-four previously characterized strains of Haemophilus influenzae were used to evaluate the specificity of previously published SNP PCR primers for the detection of the N526K substitution in PBP3 of BLNAR isolates using real-time PCR. Hasegawa et al. primers that amplify strains without a substitution at 526 and fail to amplify strains with N526K were 100% sensitive and specific for detecting N526K. However, primer sets of Hasegawa et al. and Nakamura et al. designed to amplify strains with N526K, but not strains without a substitution, were unable to do this reliably because the primers were specific for N526K encoded by AAG and failed to amplify strains with N526K encoded by AAA. A review of N526K strains deposited on GenBank revealed an even distribution of AAG and AAA codons for N526K in European and Australian BLNAR strains, whereas only the AAG codon was seen in Japanese strains. The exclusive presence of the AAG codon in Japanese strains appears to be independent of the use of the SNP PCR primers evaluated here and remains unexplained.

Prevalence and genotypic characteristics of β-lactamase-negative ampicillin-resistant Haemophilus influenzae in Australia

To determine the prevalence of β-lactamase-negative ampicillin-resistant (BLNAR) Haemophilus influenzae in Australia and characterize the associated amino acid substitutions in penicillin-binding protein 3. Two hundred consecutive non-repeat clinical isolates of H. influenzae were collected and β-lactamase-negative isolates were screened for reduced ampicillin susceptibility using an ampicillin 2 μg disc (breakpoint <17 mm) and Etest (breakpoint ≥0.25 mg/L). All screen-positive isolates had their ampicillin MICs determined by reference broth microdilution and their ftsI genes were sequenced. No BLNAR strains (MIC ≥4 mg/L) were found, but 5 (2.5%) low BLNAR (L-BLNAR) strains (MIC ≥2 mg/L) and 36 (18%) genetic BLNAR (gBLNAR) strains (R517H or N526K) were found. Of the gBLNAR strains, four had the R517H substitution and the remainder had N526K, while no strains had combined N526K and M377I/S385T/L389F substitutions. A number of strains with neither R517H nor N526K substitutions that did not meet the gBLNAR definition had other BLNAR-associated substitutions. BLNAR and L-BLNAR strains are uncommon in Australia, while gBLNAR strains are more common than previously recognized.

Polymorphism in ftsI gene and -lactam susceptibility in Portuguese Haemophilus influenzae strains: clonal dissemination of -lactamase-positive isolates with decreased susceptibility to amoxicillin/clavulanic acid

The aim of this study was to characterize ampicillin resistance mechanisms in clinical isolates of Haemophilus influenzae from Portugal. Association between specific patterns of amino acid substitutions in penicillin-binding protein 3 (PBP3) (with or without β-lactamase production) and β-lactam susceptibility as well as genetic relatedness among isolates were investigated. Two-hundred and forty non-consecutive H. influenzae isolates chosen according to their different ampicillin MICs [101 β-lactamase-non-producing ampicillin-resistant (BLNAR) isolates, 80 β-lactamase-producing ampicillin-resistant (BLPAR) isolates and 59 β-lactamase-non-producing ampicillin-susceptible (BLNAS) isolates] were analysed. The β-lactamase-encoding bla(TEM-1) gene was detected by PCR. The ftsI gene encoding PBP3 was sequenced. Genetic relatedness among isolates was examined by PFGE. Of the 240 H. influenzae isolates, 141 had mutations in the transpeptidase domain of the ftsI gene, including most BLNAR strains (94/101, 93.1%) and a high percentage of BLPAR strains (47/80, 58.8%). As previously reported, the latter have been described as β-lactamase-positive amoxicillin/clavulanic acid resistant (BLPACR). The most common amino acid substitutions were identified near the KTG motif: N526K (136/141, 96.5%), V547I (124/141, 87.9%) and N569S (121/141, 85.8%). The 141 strains were divided into 31 ftsI mutation patterns and included six groups (I, IIa, IIb, IIc, IId and III-like). BLNAR strains were genetically diverse but close genetic relationships were demonstrated among BLPACR strains. This study shows that the non-enzymatic mechanism of resistance to β-lactams is widespread among H. influenzae isolates in Portugal. Clonal dissemination of BLPACR strains showing high resistance to ampicillin and reduced susceptibility to amoxicillin/clavulanic acid was documented.

Antimicrobial Resistance in Haemophilus influenzae Respiratory Tract Isolates in Korea: Results of a Nationwide Acute Respiratory Infections Surveillance

Antimicrobial susceptibility patterns and beta-lactam resistance mechanisms of 544 Haemophilus influenzae isolates through the nationwide Acute Respiratory Infections Surveillance (ARIS) network in Korea during 2005 and 2006 were determined. Resistance to ampicillin was 58.5%, followed by resistance to cefuroxime (23.3%), clarithromycin (18.7%), cefaclor (17.0%), amoxicillin-clavulanate (10.4%), and chloramphenicol (8.1%). Levofloxacin and cefotaxime were the most active agents tested in this study. beta-Lactamase production (52.4%) was the main mechanism of ampicillin resistance, affecting 96.1% of TEM-1-type beta-lactamase. According to their beta-lactam resistance mechanisms, all isolates were classified into the following groups: beta-lactamase-negative, ampicillin-sensitive (BLNAS) strains (n = 224; 41.5%); beta-lactamase-positive, ampicillin-resistant (BLPAR) strains (n = 255; 47.2%); beta-lactamase-negative, ampicillin-resistant (BLNAR) strains (n = 33; 6.1%); and beta-lactamase-positive, amoxicillin-clavulanate-resistant (BLPACR) strains (n = 28; 5.2%). Among the BLNAR and BLPACR strains, there were various patterns of multiple-amino-acid substitutions in penicillin-binding protein 3. Particularly, among BLNAR, group III isolates, which had three simultaneous substitutions (Met377Ile, Ser385Thr, and Leu389Phe), were identified for the first time in Korea. Three group III strains displayed the highest MIC of cefotaxime (1 to 2 mug/ml). The results indicate the importance of monitoring a changing situation pertaining to the increase and spread of BLNAR and BLPACR strains of H. influenzae for appropriate antibiotic therapy for patients with respiratory tract infections in Korea.

Canadian β-lactamase–negative Haemophilus influenzae isolates showing decreased susceptibility toward ampicillin have significant penicillin binding protein 3 mutations

Nineteen β-lactamase–negative ampicillin-resistant (BLNAR) and 2 β-lactamase–positive amoxicillin-clavulanic acid–resistant Canadian Haemophilus influenzae strains were characterized. All 21 isolates were found to have the N526K mutations in their ftsI genes, and their ampicillin MIC 50 values were 4–8 times that of β-lactamase–negative ampicillin-susceptible strains. The difficulty in detection of BLNAR strains was discussed.

Low β-Lactamase-Negative Ampicillin-Resistant Haemophilus influenzae Strains Are Best Detected by Testing Amoxicillin Susceptibility by the Broth Microdilution Method

Ampicillin resistance in Haemophilus influenzae due to alterations in penicillin-binding proteins (beta-lactamase negative ampicillin resistant [BLNAR]) is acquiring increasing clinical and epidemiological importance. BLNAR strains with low ampicillin MICs (0.5 to 4 microg/ml) represent the majority of this population in Europe and the United States, but separating them from susceptible isolates is challenging. To investigate the best method to identify low-BLNAR strains, we studied the antibiotic susceptibilities of 94 clinical isolates of H. influenzae by microdilution, Etest, and disk diffusion: 25 had no resistance mechanisms (gBLNAS), 34 had mutations in the ftsI gene only (gBLNAR), 20 were beta-lactamase producers only (gBLPAR), and 15 showed beta-lactamase production and mutations in the ftsI gene (gBLPACR). By current CLSI breakpoints, most gBLNAR isolates were ampicillin susceptible by microdilution (76.5%) or by Etest (88.2%). Most gBLNAR strains (79.4%) were nonsusceptible to amoxicillin (the most widely used community antibiotic in the United States and Europe) when tested by microdilution. By Etest, 15% of beta-lactamase-positive isolates were nonresistant to ampicillin or amoxicillin. The poorest agreement between Etest and microdilution results was for the gBLPAR isolates (25% for ampicillin, 15% for amoxicillin, and 10% for cefaclor). Low-strength disks of ampicillin and amoxicillin-clavulanic acid poorly identified low-BLNAR isolates and are not recommended as a screening method. We suggest new amoxicillin breakpoints for BLNAR isolates as follows: susceptible, MIC < or = 0.5 microg/ml (no resistance mechanisms; pharmacokinetic/pharmacodynamic [PK/PD] data favorable); intermediate, MICs = 1 to 2 microg/ml (resistance mechanisms present but PK/PD data favorable), and resistant, MICs > or = 4 microg/ml (resistance mechanisms present and PK/PD data unfavorable).

An amino acid substitution in PBP-3 in Haemophilus influenzae associate with the invasion to bronchial epithelial cells

Haemophilus influenzae is a common pathogen of respiratory infections. We examined whether beta-lactamase-negative ampicillin-resistant (BLNAR) strains that are known to have ampicillin resistance due to a substitution of amino acid of penicillin binding protein (PBP)-3, differ from beta-lactamase-negative ampicillin-susceptible strains with regard to invasion of bronchial epithelium. After 3h incubation of each of 34 beta-lactamase-negative ampicillin-susceptible and 57 BLNAR strains in the presence of BEAS-2B cells, a human bronchial epithelium cell line, extracellular bacteria were killed using gentamicin and intracellular bacteria numbered. All nine strains in which the efficiency of invasion was 1% or higher were BLNAR strains. The rate of invasion was significantly greater in strains with PBP-3 amino acid substitution (Met377 to Ile, Ser385 to Thr, Leu389 to Phe, and Asn526 to Lys) (n=34) than in those with no amino acid substitution. Electron microscopy showed that high invasive BLNAR strains were observed in cytoplasm of BEAS-2B cell layer. The injured cells were 9.44+/-1.76% among attaching cells examined by trypan blue staining after 6h. These data may suggest that the amino acid substitution of the PBP in BLNAR strains may at least partly play roles in macropinocytosis, leading to the invasion and injury to epithelial cells.

Comparative efficacies of different antibiotic treatments to eradicate nontypeable Haemophilus influenzaeinfection

BackgroundNonencapsulated and nontypeable Haemophilus influenzae (NTHi) is a major cause of human respiratory tract infections. Some strains of NTHi can cause invasive diseases such as septicemia and meningitis, even if H. influenzae is not generally considered to be an intracellular pathogen. There have been very few reports about the therapeutic efficacy of antibiotics against respiratory tract infection caused by NTHi in mice because it is difficult for H. influenzae to infect mice. Therefore, we evaluated the efficacy of antibiotics against NTHi in both a cell culture model and a mouse model of infection.MethodsWe used six strains of NTHi isolated from adult patients with chronic otitis media, namely three β-lactamase-negative ampicillin (AMP)-resistant (BLNAR) strains and three β-lactamase-negative AMP-susceptible (BLNAS) strains, to evaluate the efficacy of AMP, cefcapene (CFPN), levofloxacin (LVX), clarithromycin (CLR), and azithromycin (AZM) in both a cell culture infection model and a mouse infection model. In the cell culture infection model, strains that invade A549 human alveolar epithelial cells were treated with each antibiotic (1 μg/ml). In the mouse infection model, female C57BL/6 mice were intraperitoneally injected with cyclophosphamide (200 mg/kg) three days before intranasal infection with 1 × 109 colony-forming units (CFU) of NTHi and on the day of infection. After infection, the mice were orally administered each antibiotic three times daily for three days, except for AZM, which was administered once daily for three days, at a dose of 100 mg/kg/day.ResultsIn the cell culture infection model, it was found that two BLNAR strains were able to enter the cell monolayers by the process of macropinocytosis, and treatment with LVX yielded good bactericidal activity against both strains inside the cells. In the mouse infection model, no bacteria were detected by means of plating the lung homogenates of LVX-treated mice at day 4 after infection, while more than 105 CFU of bacteria per tissue sample were detected in nontreated mice.ConclusionOur findings show the outcome and rich benefits of fluoroquinolone treatment of respiratory infections caused by either invasive or noninvasive BLNAR strains of NTHi.

Prevalence of Haemophilus influenzae with resistant genes isolated from young children with acute lower respiratory tract infections in Nha Trang, Vietnam

Our study was undertaken to investigate the characteristics of Haemophilus influenzae in young children with acute lower respiratory tract infections in Nha Trang, Vietnam. The study population consisted of 116 children less than 5 years of age admitted to Khanh Hoa General Hospital due to acute lower respiratory tract infections between July 2004 and April 2005. Organisms could be detected from nasopharyngeal swabs (NP) in 72 (62.1%) of the 116 children. Haemophilus influenzae was the most common organism, and 39 strains were isolated from 39 children aged 2 to 60 months (mean age, 16 months). We examined 37 of these 39 H. influenzae strains. The serotypes of the 37 isolates were all nontypeable, and 22 strains (59.5%) were beta-lactamase producing. Polymerase chain reaction (PCR) analysis to identify resistance genes revealed that 17 strains had the TEM-1-type beta-lactamase gene alone, 6 strains had the ftsI gene with the same substitution as that in g low-beta-lactamase-negative ampicillin-resistant (g low-BLNAR) strains, and 6 strains had both the TEM-1-type beta-lactamase gene and the ftsI gene with the same substitution as that in g beta-lactamase-producing amoxicillin clavulanic acid-resistant (g BLPACR I) strains, although no BLNAR strains were found. Molecular typing by pulsed-field gel electrophoresis (PFGE) showed that the 6 g low-BLNAR strains had five PFGE patterns and the 6 g BLPACR I strains had four PFGE patterns. Our results indicate that BLNAR strains are still not prevalent, but that g low-BLNAR and g BLPACR I strains are potentially spreading in Nha Trang, Vietnam.

Clinical bacteriology and immunology in acute otitis media in children

Acute otitis media (AOM) is the most common disease seen in childhood. Streptococcus pneumoniae, non-typeable Haemophilus influenzae (NTHi), and Moraxella catarrhalis are the most frequent pathogens of all AOM episodes. The high prevalence of drug-resistant pathogens such as penicillin-resistant S. pneumoniae (PRSP) and betalactamase producing or nonproducing ampicillin-resistant H. influenzae (BLPAR or BLNAR) is causing serious clinical problems worldwide. PRSP and BLNAR have become important risk factors for intractable clinical outcome of AOM. PRSP causes a three times higher incidence of intractable AOM than susceptible strains. BLNAR strains show penicillin-binding protein gene mutation and are not only resistant to ampicillin, but also have reduced susceptibility to cephalosporin. The resistant H. influenzae pathogen has shown clonal dissemination in Japan in ways different from those of penicillin-resistant S. pneumoniae. Protection against AOM due to these pathogens may depend on pathogen-specific antibodies. Pneumococcal capsular polysaccharides (PCPs) are type specific and poorly immunogenic in children younger than 2 years old. Approximately 50% of otitis-prone children showed subnormal levels of anti-PCP IgG2 antibody. In our immunological study in children with otitis media, however, otitis-prone children were not unusually vulnerable to infections except those resulting in otitis media. This fact seems to refute the presence of a broad immunological deficit in these children. Some pathogen-specific antibodies may be directed against protein immunogens such as pneumococcal surface protein A (PspA) of S. pneumoniae, P6 of NTHi, and UspA of M. catarrhalis. The levels of antibody to P6 of NTHi in healthy children were significantly higher than those in the otitis-prone children after the age of 18 months. In general, individual antibody levels in otitis-prone individuals did not have an age-dependent rise. The failure to develop a good antibody response to common antigens such as PspA and P6 may enable the pathogen to cause persistent or recurrent disease.