You are here: Home Research Themes Antimicrobial Resistance

Antimicrobial Resistance

Article Reference Appropriate treatment of acute otitis media in the era of antibiotic resistance.
The outcome of treatment for acute otitis media (AOM) differs between various antibiotic drugs. Outcome depends upon the drugs' pharmacokinetics, but in the case of infectious diseases also on the susceptibility of the organism and the interaction between the drug and the organisms at the specific site of infection (pharmacodynamics). In the era of antibiotic resistance, it is thus important to understand the pharmacokinetics/pharmacodynamics of the various available drugs in the context of AOM and its main two pathogens, Streptococcus pneumoniae and non-typeable Haemophilus influenzae. In terms of clinical outcome, it is also important to realize that AOM is a self-limiting disease in most cases, so that response to treatment is always compared with the expected background response when not treated. A favourable clinical outcome (cure/improvement) at the end of the treatment period is expected for those in whom the pathogens are eradicated within 3-5 days, thus clinical failure rates are several fold lower in children with early eradication (within 3-5 days) compared with those in whom no early eradication takes place. Because of the higher spontaneous bacterial elimination this might not always be appreciated. In this review, the relationship between antibiotic resistance, the various antibiotic drugs and their pharmacokinetic/pharmacodynamic patterns, the bacteriological outcome and clinical outcomes are addressed. This review is meant to assist the clinician in both a better understanding of the current recommendations for the treatment of AOM and the steps to be taken to follow AOM patients.
Article Reference Urinary tract infection caused by nontypable Haemophilus influenzae in the elderly.
Article Reference Nosocomial outbreak of biotype I, multidrug-resistant, serologically non-typeable Haemophilus influenzae in a respiratory care ward in Taiwan.
Article Reference Current management of pediatric acute otitis media.
Acute otitis media (AOM) is the most common childhood bacterial infection for which antibiotics are prescribed worldwide. The most common pathogens causing AOM in children are Streptococcus pneumoniae, nontypeable Haemophilus influenzae, Moraxella catarrhalis and Group A streptococcus. Antibiotic resistance is increasing among the bacterial pathogens causing AOM, with percentages of penicillin- and macrolide-resistant S. pneumoniae strains estimated to be between 30 and 70%, and of beta-lactamase-producing H. influenzae ranging between 20 and 40%. The introduction of the seven-valent pneumococcal conjugated vaccine had a major role in decreasing the number of vaccine-related S. pneumoniae AOM episodes, recurrent AOM cases and cases requiring the insertion of ventilation tubes. In parallel, it caused a rapid shift in the microbiology of AOM, characterized by an increase in the number of non-vaccine S. pneumoniae serotypes and H. influenzae isolates. The management of AOM in childhood has evolved considerably during recent years as a result of the new insights provided by the publication of the American Academy of Pediatrics and American Academy of Family Physicians guidelines for the treatment of AOM. The new treatment guidelines establish a clear hierarchy among various antibacterials used in the treatment of AOM and also the use of an age-stratified approach to AOM by recommending an observation strategy ('watchful waiting') without the use of antibacterials for some groups of AOM patients. Adherence to such a policy in patients with uncertain/questionable AOM diagnosis and/or mild-to-moderate symptoms, in addition to its implementation in patients over 2 years of age, could substantially reduce the use of antibacterials for the treatment of AOM and play a major role in the strategy of decreasing antibacterial resistance.
Article Reference Binding of complement regulators to invasive nontypeable Haemophilus influenzae isolates is not increased compared to nasopharyngeal isolates, but serum resistance is linked to disease severity.
The aim of the present study was to analyze the importance of nontypeable Haemophilus influenzae (NTHi) isolated from patients with sepsis (invasive isolates) compared to nasopharyngeal isolates from patients with upper respiratory tract infection for resistance to complement-mediated attack in human serum and to correlate this result with disease severity. We studied and characterized cases of invasive NTHi disease in detail. All patients with invasive NTHi isolates were adults, and 35% had a clinical presentation of severe sepsis according to the ACCP/SCCM classification of sepsis grading. Moreover, 41% of the patients had evidence of immune deficiency. The different isolates were analyzed for survival in human serum and for binding of 125I-labeled, purified human complement inhibitors C4b-binding protein (C4BP), factor H, and vitronectin, in addition to binding of regulators directly from serum. No significant differences were found when blood-derived and nasopharyngeal isolates were compared, suggesting that interactions with the complement system are equally important for NTHi strains, irrespective of isolation site. Interestingly, a correlation between serum resistance and invasive disease severity was found. The ability to resist the attack of the complement system seems to be important for NTHi strains infecting the respiratory tract as well as the bloodstream.
Article Reference Haemophilus ducreyi SapA contributes to cathelicidin resistance and virulence in humans.
Haemophilus ducreyi is an extracellular pathogen of human epithelial surfaces that resists human antimicrobial peptides (APs). The organism's genome contains homologs of genes sensitive to antimicrobial peptides (sap operon) in nontypeable Haemophilus influenzae. In this study, we characterized the sap-containing loci of H. ducreyi 35000HP and demonstrated that sapA is expressed in broth cultures and H. ducreyi-infected tissue; sapA is also conserved among both class I and class II H. ducreyi strains. We constructed a nonpolar sapA mutant of H. ducreyi 35000HP, designated 35000HPsapA, and compared the percent survival of wild-type 35000HP and 35000HPsapA exposed to several human APs, including alpha-defensins, beta-defensins, and the cathelicidin LL-37. Unlike an H. influenzae sapA mutant, strain 35000HPsapA was not more susceptible to defensins than strain 35000HP was. However, we observed a significant decrease in the survival of strain 35000HPsapA after exposure to LL-37, which was complemented by introducing sapA in trans. Thus, the Sap transporter plays a role in resistance of H. ducreyi to LL-37. We next compared mutant strain 35000HPsapA with strain 35000HP for their ability to cause disease in human volunteers. Although both strains caused papules to form at similar rates, the pustule formation rate at sites inoculated with 35000HPsapA was significantly lower than that of sites inoculated with 35000HP (33.3% versus 66.7%; P = 0.007). Together, these data establish that SapA acts as a virulence factor and as one mechanism for H. ducreyi to resist killing by antimicrobial peptides. To our knowledge, this is the first demonstration that an antimicrobial peptide resistance mechanism contributes to bacterial virulence in humans.
Article Reference Characterization of nontypeable Haemophilus influenzae collected from respiratory infections and invasive disease cases in Manitoba, Canada.
With the introduction of the Haemophilus influenzae serotype b (Hib) vaccine, invasive Hib disease has decreased substantially, but nontypeable H. influenzae (NT Hi) disease appears to be increasing. In order to understand the origin of NT Hi strains and their relationship with serotypeable strains, we analysed 125 NT Hi isolates collected from individual patients with either invasive disease (70 isolates) or respiratory tract infections (55 isolates). Serotype-specific and capsular transport genes were absent by PCR analysis, confirming their nonencapsulated status, which also suggested the NT Hi isolates were not encapsulated strains that shed their capsules. Multilocus sequence typing confirmed the NT Hi isolates did not have the same genetic background as serotypeable strains, including Hib. Despite the genetic heterogeneity found, two major genetic clusters were identified, both containing invasive and respiratory isolates. Fourteen invasive isolates and nine respiratory isolates produced beta-lactamase and were ampicillin resistant. More invasive (26.8%) than respiratory isolates (10.9%) showed decreased susceptibility towards ampicillin by a mechanism unrelated to beta-lactamase production. Besides a change in the capsule status of invasive Hi strains, the burden of invasive Hi disease, which used to be mainly a childhood disease, has now shifted to involve both adults and infants.
Article Reference Chronic obstructive pulmonary disease: role of bacteria and updated guide to antibacterial selection in the older patient.
Chronic obstructive pulmonary disease (COPD) remains a major cause of morbidity and mortality worldwide. COPD is especially prevalent in the elderly, affecting 25% of those aged>or=75 years. The course of the disease in the elderly is often complicated by co-morbid conditions, and its management is complicated by drug-drug interactions. Exacerbations of COPD increase rates of hospitalization and mortality and decrease quality of life. Exacerbations are marked by an increase from baseline in dyspnoea, sputum volume and sputum purulence. Approximately 50% of acute exacerbations of symptoms in COPD are caused by non-typeable Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae and Pseudomonas aeruginosa. Stratification of exacerbations based on severity of symptoms and signs, and severity of underlying COPD, is useful in selecting patients likely to benefit from antibacterial therapy. Patients who are hospitalized with exacerbations, those who have all three symptoms (increased dyspnoea, sputum volume and sputum purulence), and those with severe underlying COPD and exacerbations benefit most from antibacterials. Antibacterial susceptibility patterns among the bacterial pathogens are evolving, and knowledge of local susceptibility patterns is useful in antibacterial selection. Penicillin, amoxicillin, cotrimoxazole (trimethoprim/sulfamethoxazole) and doxycycline should not be used as an initial antibacterial because of resistance patterns. We recommend second-/third-generation cephalosporins, amoxicillin/clavulanic acid, azithromycin and respiratory fluoroquinolones as initial choices. In patients at risk of colonization by, and infection as a result of, P. aeruginosa, ciprofloxacin, levofloxacin or an advanced penicillin/penicillinase combination effective against this species should be used. Drug-drug interactions should be considered in antibacterial choice. The goals of antibacterial therapy for exacerbations of COPD are the prevention of complications such as respiratory failure and death, and the reduction of treatment failures. The role of pathogenic bacteria in progression of stable COPD and the use of prophylactic antibacterials in stable COPD are under investigation. Currently available evidence does not support routine clinical use of prophylactic antibacterials in stable COPD. In conclusion, pathogenic bacteria cause a significant proportion of acute exacerbations of COPD. Use of antibacterials, based on current susceptibility patterns, is beneficial in patients with severe COPD experiencing exacerbations and in patients with severe exacerbations.
Article Reference Otitis media: viruses, bacteria, biofilms and vaccines.
Otitis media typically presents as either acute otitis media (AOM), with symptoms including fever, otalgia, otorrhoea or irritability and short duration; or as otitis media with effusion (OME), which is often asymptomatic and characterised by accumulation of fluid in the middle ear. Diagnostic certainty of otitis media is challenging, given the young age of patients and variability of symptoms. Otitis media predominantly occurs as coincident to viral upper respiratory tract infections and/or bacterial infections. Common viruses that cause upper respiratory tract infection are frequently associated with AOM and new-onset OME. These include respiratory syncytial virus, rhinovirus, adenovirus, parainfluenza and coronavirus. Predominant bacteria that cause otitis media are Streptococcus pneumoniae, Moraxella catarrhalis, and non-typeable Haemophilus influenzae. Antibiotic therapy does not significantly benefit most patients with AOM, but long-term prophylactic antibiotic therapy can reduce the risk of otitis media recurrence among children at high risk. In Australia, 84% of AOM is treated with antibiotic therapy, which contributes to development of antibiotic resistance. Vaccine development is a key future direction for reducing the world burden of otitis media, but requires polymicrobial formulation and ongoing monitoring and modification to ensure sustained reduction in disease burden.
Article Reference Nontypeable Haemophilus influenzae protein E binds vitronectin and is important for serum resistance.
Nontypeable Haemophilus influenzae (NTHi) commonly causes local disease in the upper and lower respiratory tract and has recently been shown to interfere with both the classical and alternative pathways of complement activation. The terminal pathway of the complement system is regulated by vitronectin that is a component of both plasma and the extracellular matrix. In this study, we identify protein E (PE; 16 kDa), which is a recently characterized ubiquitous outer membrane protein, as a vitronectin-binding protein of NTHi. A PE-deficient NTHi mutant had a markedly reduced survival in serum compared with the PE-expressing isogenic NTHi wild type. Moreover, the PE-deficient mutant showed a significantly decreased binding to both soluble and immobilized vitronectin. In parallel, PE-expressing Escherichia coli bound soluble vitronectin and adhered to immobilized vitronectin compared with controls. Surface plasmon resonance technology revealed a K(D) of 0.4 microM for the interaction between recombinant PE and immobilized vitronectin. Moreover, the PE-dependent vitronectin-binding site was located at the heparin-binding domains of vitronectin and the major vitronectin-binding domain was found in the central core of PE (aa 84-108). Importantly, vitronectin bound to the surface of NTHi 3655 reduced membrane attack complex-induced hemolysis. In contrast to incubation with normal human serum, NTHi 3655 showed a reduced survival in vitronectin-depleted human serum, thus demonstrating that vitronectin mediates a protective role at the bacterial surface. Our findings show that PE, by binding vitronectin, may play an important role in NTHi pathogenesis.
Article Reference [Peritonitis in the course of peritoneal dialisis caused by Haemophilus influenzae with BLNAR phenotype].
Most common bacterial species causing peritonitis in the course of peritoneal dialysis (PDP) are coagulase-negative staphylococci, Staphylococcus aureus and streptococci. Haemophilus influenzae is rarely associated with PDP. Hereby we present the first known case of APD-associated peritonitis caused by non-type able H. influenzae (NTHi) presenting the beta-lactamase negative, ampicillin-resistant (BLNAR) phenotype. An 18 year old boy who had been treated with the APD for 12 months due to SLE was admitted in good general condition with diagnosis of PDP. Standard diagnostic and therapeutical procedures were initiated. Dialysis fluid was turbid with cytosis of 435 WBC/ml. From dialysis fluid pure culture of Gram-negative coccobacillus was isolated. The isolate was identified as a BLNAR phenotype. The same bacterium was isolated from nasal swab. Blood cultures were negative. After evaluation of antimicrobial susceptibility the treatment was changed for the oral ciprofloxacin. The treatment was successful. Control tests 2 days later revealed cytosis of 15 WBC/mm3 and control cultures of peritoneal fluid were negative. After two weeks of treatment the patient was discharged in a good condition. Haemophilus influenzae is a bacterium frequently colonizing the nasopharyngeal cavity. A PCR-based method allowed to classify isolates as NTHi. Infection was probably of the respiratory origin as the isolates (from peritoneal fluid and nasal swab) were undistinguishable. There are only few reports describing this species as an ethiologic agent of peritonitis. This case prove that Haemophilus species should be taken into account as a possible aethiologic agent of PDP, especially in patients on immunosupression with carrier state of H. influenzae in the upper respiratory tract. This kind of microorganism requires specific conditions during its growing in vitro. Identification of its sensitivity to antibiotics is essential in order to detect strains of BLNAR phenotype, as it is a crucial part of an effective antibiotic therapy.
Article Reference Intercellular adhesion and biocide resistance in nontypeable Haemophilus influenzae biofilms.
Respiratory infections caused by nontypeable Haemophilus influenzae (NTHi) are a major medical problem. Evidence suggests that the ability to form biofilms on mucosal surfaces may play a role in NTHi pathogenesis. However, the factors that contribute to NTHi biofilm cohesion remain largely unknown. In this study we investigated the biofilm growth and detachment phenotypes of eight NTHi clinical strains in vitro. We found that the majority of strains produced biofilms within 6h when cultured statically in tubes. Biofilm formation was inhibited when culture medium was supplemented with proteinase K or DNase I. Both enzymes also caused significant detachment of pre-formed NTHi biofilms. These findings indicate that both proteinaceous adhesins and extracellular DNA contribute to NTHi biofilm cohesion. Treatment of NTHi biofilms cultured in centrifugal filter devices with DNase I, but not with proteinase K, caused a significant decrease in fluid convection through the biofilms. These results suggest that extracellular DNA is the major volumetric component of the NTHi biofilm matrix. Mechanical or enzymatic disruption of NTHi biofilms cultured in microtiter plates significantly increased their sensitivity to killing by SDS, cetylpyridinium chloride, chlorhexidine gluconate, povidone iodine and sodium hypochlorite. These findings indicate that biocide resistance in NTHi biofilms is mediated to a large part by the cohesive and protective properties of the biofilm matrix. Understanding the mechanisms of biofilm cohesion and biocide resistance in NTHi biofilms may lead to new methods for treating NTHi-associated infections.
Article Reference Lex2B, a phase-variable glycosyltransferase, adds either a glucose or a galactose to Haemophilus influenzae lipopolysaccharide.
Nontypeable Haemophilus influenzae is a commensal that frequently causes otitis media and respiratory tract infections. The lex2 locus encodes a glycosyltransferase that is phase variably expressed and contributes to the significant intrastrain heterogeneity of lipopolysaccharide (LPS) composition in H. influenzae. In serotype b strains, Lex2B adds the second beta-glucose in the oligosaccharide extension from the proximal heptose of the triheptose inner core backbone; this extension includes a digalactoside that plays a role in resistance of the bacteria to the killing effect of serum. As part of our studies of the structure and genetics of LPS in nontypeable H. influenzae, we show here that there are allelic polymorphisms in the lex2B sequence that correlate with addition of either a glucose or a galactose to the same position in the LPS molecule across strains. Through exchange of lex2 alleles between strains we show that alteration of a single amino acid at position 157 in Lex2B appears to be sufficient to direct the alternative glucosyl- or galactosyltransferase activities. Allelic exchange strains express LPS with altered structure and biological properties compared to the wild-type LPS. Thus, Lex2B contributes to both inter- and intrastrain LPS heterogeneity through its polymorphic sequences and phase-variable expression.
Article Reference Susceptibilities of Haemophilus influenzae, Streptococcus pneumoniae, including serotype 19A, and Moraxella catarrhalis paediatric isolates from 2005 to 2007 to commonly used antibiotics.
The aim of this study was to evaluate susceptibility to common paediatric antibiotics for Streptococcus pneumoniae, non-typeable Haemophilus influenzae and Moraxella catarrhalis isolated from 2005 through 2007.
Article Reference 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.
Article Reference Lipooligosaccharides containing phosphorylcholine delay pulmonary clearance of nontypeable Haemophilus influenzae.
Nontypeable Haemophilus influenzae (NTHi) causes pulmonary infections in patients with chronic obstructive pulmonary disease and other mucociliary clearance defects. Like many bacteria inhabiting mucosal surfaces, NTHi produces lipooligosaccharide (LOS) endotoxins that lack the O side chain. Persistent NTHi populations express a discrete subset of LOS glycoforms, including those containing phosphorylcholine (PCho). In this study, we compared two NTHi strains with isogenic mutants lacking PCho for clearance from mice following pulmonary infection. Consistent with data from other model systems, populations of the strains NTHi 2019 and NTHi 86-028NP recovered from mouse lung contained an increased proportion of PCho+ variants compared to that in the inocula. PCho- mutants were more rapidly cleared. Serial passage of NTHi increased both PCho content and bacterial resistance to clearance, and no such increases were observed for PCho- mutants. Increased PCho content was also observed in NTHi populations within non-endotoxin-responsive C3H/HeJ and Toll-like receptor 4 null (TLR4-/-) mice, albeit at later times postinfection. Changes in bacterial subpopulations and clearance were unaffected in TLR2-/- mice compared to the subpopulations in and clearance from mice of the parental strain. The clearance of PCho- mutants occurred at earlier time points in both strain backgrounds and in all types of mice. Comparison of bacterial populations in lung tissue cryosections by immunofluorescent staining showed sparse bacteria within the air spaces of C57BL/6 mice and large bacterial aggregates within the lungs of MyD88-/- mice. These results indicate that PCho promotes bacterial resistance to pulmonary clearance early in infection in a manner that is at least partially independent of the TLR4 pathway.
Article Reference Low rate of nasopharyngeal carriage and high rate of ampicillin resistance for Haemophilus influenzae among healthy children younger than 5 years old in northern Taiwan.
Surveillance data of colonization by Haemophilus influenzae in Taiwan are lacking. This study aimed to define the nasopharyngeal carriage rate of H. influenzae among children younger than 5 years in northern Taiwan, and to determine the antibiotic susceptibility, serotype and the clonal relationship of these isolates.
Article Reference Mouse models for the study of mucosal vaccination against otitis media.
Otitis media (OM) is one of the most common infectious diseases in humans. The pathogenesis of OM involves nasopharyngeal (NP) colonization and retrograde ascension of the pathogen up the Eustachian tube into the middle ear (ME). Due to increasing rates of antibiotic resistance, there is an urgent need for vaccines to prevent infections caused by the most common causes of bacterial OM, including nontypeable Haemophilus influenzae, Streptococcus pneumoniae and Moraxella catarrhalis. Current vaccine strategies aim to diminish bacterial NP carriage, thereby reducing the likelihood of developing acute OM. To be effective, vaccination should induce local mucosal immunity both in the ME and in the NP. Studies in animal models have demonstrated that the intranasal route of vaccination is particularly effective at inducing immune responses in the nasal passage and ME for protection against OM. The mouse is increasingly used in these models, because of the availability of murine reagents and the existence of technology to manipulate murine models of disease immunologically and genetically. Previous studies confirmed the suitability of the mouse as a model for inflammatory processes in acute OM. Here, we discuss various murine models of OM and review the applicability of these models to assess the efficacy of mucosal vaccination and the mechanisms responsible for protection. In addition, we discuss various mucosal vaccine antigens, mucosal adjuvants and mucosal delivery systems.
Article Reference Antimicrobial effect of fluoroquinolones for the eradication of nontypeable Haemophilus influenzae isolates within biofilms.
Biofilms can be defined as communities of microorganisms attached to a surface. Those bacterial biofilms cause serious problems, such as antibiotic resistance and medical device-related infections. Nontypeable Haemophilus influenzae (NTHi) is an important pathogen in respiratory infections, as it forms biofilms both in vitro and in vivo such as human middle ear. Recent reports indicate that otitis media, paranasal sinusitis and lower respiratory tract infections caused by Haemophilus influenzae have become more difficult to treat with oral antibiotic therapy. However, there has been no attention given to antibiotic eradication of NTHi biofilm. To investigate the antimicrobial effect of various antibiotics against NTHi biofilm formation, we conducted the following comparative study using both beta-lactamase-negative ampicillin (AMP)-susceptible (BLNAS) and AMP-resistant (BLNAR) NTHi strains. In a microtiter biofilm assay, both levofloxacin and gatifloxacin, of the fluoroquinolone antibiotic group, significantly inhibited biofilm formation by BLNAS and BLNAR NTHi in a dose-dependent fashion compared to ampicillin of the penicillin antibiotic group, cefotaxime of the cephalosporin antibiotic group, and both erythromycin and clarithromycin of the macrolide antibiotic group. Furthermore, in flow cell chamber studies, confocal laser scanning microscopy counted survival bacteria in mature biofilm had been treated with gatifloxacin, ampicillin, cefotaxime and erythromycin. Only gatifloxacin completely killed the BLNAR NTHi isolates within biofilms without regard to the thickness of biofilm formation. The results of this study suggest that fluoroquinolones potentially have a role in therapy against diseases caused by both BLNAS and BLNAR NTHi isolates within biofilms.
Article Reference Outer membrane protein analysis of ampicillin-resistant isolates of Haemophilus influenzae from Saudi Arabia.
Haemophilus influenzae isolates characterized in a previously published study from the Kingdom of Saudi Arabia were analysed by outer membrane protein (OMP) profiling. Isolates from patients with confirmed respiratory tract infections were investigated. Antibiotic susceptibility tests in vitro showed 25/129 (19.4%) had various degrees of reduced susceptibility to ampicillin although all were fully susceptible to ceftazidime and ciprofloxacin. OMP profiles of the beta-lactamase mediated ampicillin-resistant and beta-lactamase negative; ampicillin intermediate resistant strains (BLNAI) isolated were investigated. Dendrograms of scanned SDS-PAGE of these strains showed 15 different groupings from the 15 non-typable (NTHi) isolates tested demonstrating a high degree of heterogeneity whereas the 5 Hib isolates demonstrated significantly closer relatedness and were probably clonal. The present study demonstrates the groupings of H. influenzae strains by OMP profile analysis which did not correlate with the beta-lactamase production ability, BLNAI isolates, geographical origin or biotype.