Haemophilus influenzae is a small gram-negative rod or coccobacillus that is part of the normal flora of the upper respiratory tract of children and adults. It can be transmitted through contact with respiratory secretions. Haemophilus influenzae causes a broad spectrum of infections including pneumonia, bacteremia, meningitis, bone and joint infections, peritonitis, endophthalmitis, otitis media, sinusitis, cellulitis, and conjunctivitis. Invasive infection has the highest incidence among the elderly. Based on capsular polysaccharides, H. influenzae can be separated into 6 serotypes (a-f). Nonencapsulated strains will not react with typing antisera and are described as nontypeable. Before the use of H. influenzae type b (Hib) vaccine, Hib was the most common cause of bacterial meningitis in children in the United States. Urwin et al1 found, with the decline in the rate of Hib disease, that the relative importance of H. influenzae serotype f (Hif) increased, and the proportion of Hif rose from 1% in 1989 to 17% in 1994. There are no vaccines available against H. influenzae non-type b disease.
Ampicillin resistance in H. influenzae is most commonly caused by production of a TEM-type β-lactamase that inactivates ampicillin. Occasional ampicillin-resistant strains result from altered penicillin-binding proteins in β-lactamase-negative and ampicillin-resistant isolates.2,3
We were interested in determining the susceptibility patterns of H. influenzae case isolates collected from population-based active surveillance in Minnesota and in determining the incidence of β-lactam drug resistance among those isolates that do not produce β-lactamase. In addition, we were interested in describing the epidemiology of invasive H. influenzae disease in Minnesota.
The Minnesota Department of Health (MDH) began statewide (population is 5.1 million in the 2005 estimate US Census) active laboratory-based surveillance for invasive H. influenzae disease as part of the Centers for Disease Control and Prevention Emerging Infections Program Active Bacterial Core Surveillance in 1995. Both clinical and demographic case information were collected on standardized forms from health care providers and hospital-based infection control practitioners. All available case isolates were submitted to the MDH Public Health Laboratory (PHL). For this study, all cases and case isolates between January 1, 2002, and December 31, 2005, were reviewed.
Monthly or twice-monthly contacts were made with hospitals, clinics, and reference laboratories to verify reported and solicit unreported cases. Laboratory audits were conducted every 6 months in the 7-county Minneapolis/St Paul metropolitan area (approximately 50% of the state's population) and in facilities outside the 7-county Minneapolis/St Paul metropolitan area with 200 acute beds or more. A 20% random sample of the rest of the state's laboratories was reviewed annually to identify if there were missing cases.
A case of invasive H. influenzae disease was defined as an illness, with H. influenzae isolated from a normally sterile body site in a Minnesota resident. Normally, sterile body sites are defined as the following: blood, cerebrospinal fluid, pleural fluid, peritoneal fluid, pericardial fluid, joint, bone, lymph node, brain, heart, liver, spleen, vitreous fluid, kidney, pancreas, or ovary. Positive antigen detection, counter immunoelectrophoresis, or other non-culture-based methods are not sufficient case definitions for reporting in this surveillance system.
Case isolates were confirmed as H. influenzae and biotyped using urease, indole, and ornithine decarboxylase reactions. Isolates were serotyped by slide agglutination (Difco Laboratories, Detroit, Minn).
Minimum inhibitory concentrations (MICs) were determined by broth microdilution panel (PML Microbiologicals, Wilsonville, Ore) on all available case isolates collected between January 1, 2002, and December 31, 2005. Testing and interpretation were conducted in accordance with the Clinical and Laboratory Standards Institute/National Committee on Clinical Laboratory Standards guidelines.
Panels contained Haemophilus Test Medium broth and varying concentrations of 12 antibiotics: ampicillin, amoxicillin-clavulanate, azithromycin, chloramphenicol, ciprofloxacin, cefuroxime, meropenem, rifampin, trimethoprim-sulfamethoxazole, cefotaxime, tetracycline, and telithromycin. β-Lactamase testing was performed on all isolates by Nitrocefin Beta Lactamase Disk (Remel, Lenexa, Kan).
Statistical analysis of the data was performed using EpiInfo software version 6.04d (Centers for Disease Control and Prevention, Atlanta, Ga).
Two hundred seventeen cases (1.1/100,000 annually) of invasive H. influenzae were reported between January 1, 2002, and December 31, 2005, (age, newborn to 98 years) including 85 cases of pneumonia (39.2%), 71 cases of bacteremia without focus (32.7%), and 21 cases of meningitis (9.7%). Thirty-four of the cases (15.7%) were younger than 5 years, and 82 of the cases (37.8%) were 70 years old or older.
One hundred eighty-four case isolates (84.8%) were received at the MDH PHL and were confirmed as H. influenzae and serotyped (Table 1). Most (58.7%) case isolates were nontypeable.
Nine of the case isolates (4.9%) were H. influenzae serotype b; 4 were from children younger than 5 years. Of those 4, one 5-month-old and one 9-month-old had not been vaccinated for Hib; one 4-month-old and one 10-month-old had each received 1 vaccination.
Forty-two of the case isolates (22.8%) were serotype f, 6 (3.3%) were serotype a, 3 (1.6%) were serotype c, and 2 of the case isolates (1.1%) were serotype d. Serotype e was identified in 14 of the case isolates (7.6%).
Of the 42 case isolates identified as serotype f, sex was divided in half equally. Seventy-six percent of cases occurred in adults, and 17% of the cases occurred in children younger than 5 years. There were 7 cases of serotype f meningitis; 6 of these were in children younger than 5 years, all survived. Four patients with serotype f died, all were adults. Three of those 4 had pneumonia, and the other had bacteremia without another focus of infection. Two of those 4 had significant underlying disease.
Half of the 6 serotype a cases were younger than 5 years, and of those three, 2 had meningitis. All cases survived.
Twenty-five of the cases (11.5%) died. Twenty (80%) had significant underlying conditions including chronic obstructive pulmonary disease, cancer, diabetes mellitus, and heart and renal failure. Sixteen of those 25 had lived in the 7-county Minneapolis/St Paul metropolitan area. Eighteen cases presented with pneumonia and 7 with bacteremia without another focus of infection. Sixteen cases were older than 70 years, 5 were between 50 and 69 years, 2 were between 40 and 49 years, and 2 cases were newborn (gestational ages, 24 and 29 weeks). Nineteen of these 25 case isolates were nontypeable, 4 were serotype f, 1 was serotype c, and 1 was serotype e.
Of the 19 meningitis case isolates, 12 were typeable: 7 were serotype f, 2 were serotype b, 2 were serotype a, and 1 was serotype e. Most case isolates (84) had pneumonia as the type of infection. Thirty-one of these 84 were typeable: 18 were serotype f, 6 were serotype e, 4 were serotype b, 2 were serotype a, and 1 was serotype c. There were 60 case isolates with bacteremia as the main focus of infection; 17 were typeable: 7 were serotype f, 4 were serotype e, 2 were serotype c, 2 were serotype a, 1 was serotype b, and 1 was serotype d.
One hundred eighty-four case isolates were available for susceptibility testing and β-lactamase testing. All of these 184 case isolates were susceptible to amoxicillin-clavulanate, ciprofloxacin, cefotaxime, meropenem, and rifampin (Table 2). Seventy-nine of these case isolates (42.9%) were nonsusceptible to one or more of the remaining antibiotics tested.
Twenty case isolates (10.9%) were nonsusceptible to trimethoprim-sulfamethoxazole (19 were resistant, and 1 was intermediate). Interestingly, there was no resistance in 2002, 8 nonsusceptible case isolates in 2003; 7 in 2004, and 5 in 2005.
Sixty-one case isolates (33.2%) were resistant to ampicillin. All 61 ampicillin-resistant case isolates were β-lactamase positive. Three case isolates were ampicillin intermediate (MIC = 2 μg/mL), amoxicillin-clavulanate susceptible (MIC = 4 μg/mL), and β-lactamase negative; however, 2 of these 3 were cefuroxime intermediate (MIC = 8 μg/mL). Four case isolates (2.2%) were cefuroxime intermediate (MIC = 8 μg/mL), and 3 of these were β-lactamase negative.
Four case isolates were multidrug resistant (resistant to ≥3 antibiotics). One case isolate was ampicillin resistant and had an MIC of greater than 32 μg/mL to both azithromycin and telithromycin. Two case isolates were resistant to ampicillin, chloramphenicol, trimethoprim-sulfamethoxazole, and tetracycline. One case isolate was resistant to ampicillin, chloramphenicol, and tetracycline. Two of these 4 were serotype b, and 2 were nontypeable. One of the multidrug-resistant serotype b case isolates was in a 10-month-old that had received 1 vaccination.
Four ampicillin-resistant case isolates (2.2%) were from cases of meningitis. Nine of the 25 fatal cases had an isolate that was ampicillin resistant. None of the fatal cases was multidrug resistant. Of the 184 case isolates, there was no correlation between residence, sex, survival status, age, site or type of infection, or serotype, and resistance.
Campos et al4 found, through an active community surveillance study in Spain, that in the post-Hib vaccination era, most invasive H. influenzae disease infections were due to nonencapsulated strains and occurred in the extreme ages of life particularly in patients with predisposing conditions, including impaired immunity and respiratory diseases.5 Tsang et al6 studied 52 nonserotype b H. influenzae isolates from invasive disease in the province of Manitoba, Canada, during 2000-2004 and found that half of the isolates were serotype a, and 38.5% were nontypeable. A study in Brazil from March 1996 to September 2000 found that the incidence of H. influenzae serotype a meningitis increased 8 times (from 0.02 to 0.16 cases/100,000 person-years).7 During a study of H. influenzae isolates responsible for meningitis in Poland from 1997 to 2004, approximately 95.1% belonged to serotype b, and 2 isolates were serotype f. Vaccination in Poland is on a voluntary basis and not free of charge.8 Denmark studied invasive H. influenzae from 1995 to 2001. Thirteen cases were identified as serotype f. All cases were older than 55 years, and 77% were women.9 Bajanca et al10 studied invasive H. influenzae in Portugal during 1989 to 2001 and found 1 serotype f isolated for the first time.
We found that most invasive infections with H. influenzae occurred in individuals 60 years and older (50.6%) or children younger than 5 years (15.7%), and most strains were nontypeable. In children younger than 5 years, we found 17% were Hif, whereas Urwin et al1 found that 26% of invasive Hif cases occurred in children younger than 5 years. One of the greatest public health achievements of the late 20th century is the near elimination of invasive H. influenzae serotype b disease. The Hib conjugate vaccines have consistently decreased incidence of invasive disease by greater than 90% in all locations where they have been incorporated into routine infant immunization schedules.11
The first antimicrobial-resistant isolate of H. influenzae was identified in 1972 and was a β-lactamase-producing ampicillin-resistant type b H. influenzae.12 During the past 32 years, the β-lactamase-mediated ampicillin resistance in H. influenzae has become more prevalent.13 β-Lactamase production is the primary mechanism of resistance to penicillins among H. influenzae isolates.
In Minnesota, all invasive H. influenzae case isolates collected between 2002 and 2005 were susceptible to amoxicillin-clavulanate, which contains a β-lactamase inhibitor. All ampicillin-resistant case isolates during this surveillance period produced β-lactamase. Although no β-lactamase-negative case isolates were found that were ampicillin resistant, 3 isolates did exhibit an intermediate ampicillin MIC of 2 μg/mL. Overall, more than a third of the case isolates were resistant to ampicillin, and 11% of the case isolates were resistant to trimethoprim/sulfamethoxazole. No resistance to ciprofloxacin, cefotaxime, meropenem, or rifampin was detected, and little resistance to azithromycin, cefuroxime, chloramphenicol, telithromycin, and tetracycline was found. Four case isolates (2%) were resistant to 3 antibiotics or more. Ampicillin-resistant infections did not seem to differ by serotype, type of infection, or case fatality. A limitation of this project is that auditing of participating laboratories revealed that, although more than 99% of cases are reported to MDH through this active population and laboratory-based surveillance system, only 89% of case isolates were submitted to the MDH PHL.
Although we found little resistance other than ampicillin, including little multidrug resistance, we recommend that surveillance including susceptibility testing of H. influenzae isolates be monitored and performed in other geographic areas. We have observed significant increases in antimicrobial resistance in other common invasive bacteria from the same surveillance area including Streptococcus pneumoniae, group B Streptococcus and Staphylococcus aureus. In 2005, 13% of case isolates of community-associated methicillin-resistant S. aureus from a 12-site surveillance system in Minnesota were susceptible to erythromycin, 60% were susceptible to ciprofloxacin; however, 99% were susceptible to trimethoprim/sulfamethoxazole. Case isolates of invasive S. pneumoniae detected by population-based surveillance in Minnesota were 77% susceptible to each of the following: trimethoprim/sulfamethoxazole, erythromycin, and penicillin; and 94% were susceptible to amoxicillin. Case isolates of invasive group B Streptococcus detected by population-based surveillance in Minnesota were 67% susceptible to erythromycin.
Resistance may vary geographically and by setting (eg, hospital-acquired versus community-acquired). Recent hospitalization and the community prescription of antibiotics have been identified as risk factors for antibiotic resistance in H. influenzae.14 It is likely that the selective pressure of extensive antibiotic use has led to the widespread occurrence of ampicillin resistance in H. influenzae. Promotion and practice of judicious antibiotic use need to remain a priority issue among health care providers.
The authors thank Jean Rainbow, MPH, RN, Beth Shade, MT, ASCP, and Richard Danila, PhD, for their contribution in preparation of this manuscript. The authors also thank our colleagues at Centers for Disease Control and Prevention: Nancy Rosenstein-Messonnier, MD, and Chris Van Beneden, MD, MPH, and the infection control professionals and clinical microbiologists who collaborate with Minnesota Department of Health.