Acute Otitis Media Otopathogens during 2008-2010 in Rochester NY

Abstract

Background

The otopathogen distribution colonizing the nasopharynx (NP) and causing acute otitis media (AOM) is in flux following the introduction of PCV7 and will continue to change.

Methods

277 children were followed prospectively; tympanocentesis was performed during AOM and 208 NP samples were collected to compare with middle ear fluid (MEF) isolates. 863 NP samples were collected at 7 healthy visits between 6 and 30 months of age. All children received PCV7 until April 2010 when PCV13 was substituted. Multi-locus sequence typing (MLST) was used to speciate S. pneumoniae.

Results

The distribution of otopathogens in the MEF during the study time frame was stable. PCV7 serotypes of pneumococci were virtually absent. The frequency of isolation of S. pneumoniae was 26-36% compared with 28-34% for non-typeable Haemophilus influenzae (NTHi). M. catarrhalis isolation was less common, 7-18%. The proportion of S. pneumoniae that were penicillin non-susceptible was stable during the 3-years, 40-52%. All M. catarrhalis and 34% of NTHi were beta-lactamase-producing. NP isolates of otopathogens at onset of AOM included the isolate from the MEF and was dissimilar from the distribution at times of health. Sequence Types 320 and 199 of S. pneumoniae expressing serotypes 19A and 15 most often caused AOM.

Conclusions

The otopathogen distribution, antibiotic susceptibility and the diversity of strains within the S. pneumoniae species during 2008 through late 2010 were stable. NP isolation of otopathogens at onset of AOM better reflected, albeit incompletely, likely MEF isolates compared with NP isolates at times of health.

INTRODUCTION

The bacterial cause of acute otitis media (AOM), the antibiotic susceptibility of the organisms and the diversity of strains within the Streptococcus pneumoniae and Non-typeable Haemophilus influenzae (NTHi) species are dynamic. The major contributors to changes in otopathogen distribution, antibiotic susceptibility and strain variation in recent years have been the introduction of pneumococcal conjugate vaccines (PCVs) [1, 2] and antibiotic selection pressure [3, 4].

Asymptomatic nasopharyngeal (NP) colonization by S. pneumoniae and NTHi is common in young children. NP colonization precedes and is the critical step in pathogenesis of AOM [5]. Progression from a commensal to pathogenic state for S. pneumoniae and NTHi is facilitated by a viral upper respiratory infection (URI) in about 95% of cases [6,7]. Ascension of bacteria up the Eustachian tube, often with a concurrent virus, from the NP to the middle ear space allows establishment of infection in a closed space.

In three previous reports we described the dynamic changes occurring within the S. pneumoniae and NTHi species causing AOM as an apparent consequence of PCV introduction and antibiotic use for 1995 to 2003 [8], 2003 to 2006 [9] and 2006 to 2008 [10]. In those studies we reached several conclusions regarding AOM pathogen distribution, antibiotic susceptibility of the organisms and the diversity of strains within the S. pneumoniae and NTHi species. (1) Persistent AOM and AOM treatment failures decreased in frequency after the introduction of high dose amoxicillin therapy in 1998-2000 and PCV7 vaccination in 2001. Proportionally, NTHi became the predominant pathogen of persistent AOM and AOM treatment failures. Fewer S. pneumoniae AOM isolates were penicillin non-susceptible and more NTHi were beta-lactamase-producing [8]. (2) Three to six years after the introduction of the PCV7 vaccine, we observed in our practice a multidrug-resistant strain of S. pneumoniae of serotype 19A in nine children that caused persistent AOM. Identification of this organism by tympanocentesis and highly selective use of levofloxacin proved successful to treat the infections. The identification of this organism occurred in the context of a proportional increase in isolation of strains of S. pneumoniae not included in PCV7 and an increase in penicillin non-susceptibility among the non-PCV7 strains [9]. (3) Six to eight years after widespread use of PCV7, S. pneumoniae strains expressing vaccine-type serotypes had virtually disappeared from the NP and middle ear fluid (MEF) of vaccinated children. NP colonization and AOM had changed to non-PCV7 strains of S. pneumoniae. NTHi continued to be a major AOM pathogen. The otopathogens in first and second AOM compared with otitis prone children were found to be very similar although S. pneumoniae and NTHi were more often antibiotic resistant in the otitis prone child [10]. In this report we characterize the otopathogens causing NP colonization and AOM in children from our community, the antibiotic susceptibility of the organisms and the diversity of strains within the S. pneumoniae species for the years 2008 through October 2010. The time frame includes years 8 and 9 after introduction of PCV7 and the transition months of April to end of September 2010 when children received PCV13 in replacement of PCV7 for additional doses of PCV.Additionally, we sought to revisit that question with a focus on comparing the results of NP cultures at onset of AOM versus when children are healthy to predict the otopathogen profile causing AOM, using MEF cultures as the gold standard.

MATERIALS AND METHODS

Study Population

As previously described [10, 11], children were enrolled at 6 months of age and followed to 30 months of age; the children had no prior AOM at the time of enrollment. NP and oropharyngeal samples (hereafter for simplicity referred to as “NP samples’ for purposes of this report) were obtained at seven routine visits when the children were 6, 9, 12, 15, 18, 24, and 30 months of age. With the first and any subsequent episodes of AOM, NP cultures and middle ear fluid (MEF) collected by tympanocentesis were obtained. Antibiotic treatment regimens, outcomes of treatment and referral for insertion of tympanostomy tubes for this population has been previously described [11]

Demographic data collected included family history of AOM, daycare attendance, breast-feeding, and tobacco smoke exposure. The Rochester General Hospital IRB approved the study and written informed consent was obtained from parents before enrollment in the study.

Definition of AOM

AOM was diagnosed by pneumatic otoscopy by two of the authors (J.C., M.P.), who are both validated otoscopists, when children with acute onset of symptoms consistent with AOM had tympanic membranes (TMs) that were: (1) bulging or full, and (2) a cloudy or purulent effusion was observed, or the TM was completely opacified, and (3) TM mobility was reduced or absent.

Tympanocentesis, NP Sampling, Microbiology and Serotyping

Tympanocentesis to collect MEF for culture, NP sampling, microbiology cultures and serotyping of S. pneumoniae was done as previously described [10].

Antibiotic Susceptibility

The antibiotic susceptibility of S. pneumoniae isolates was determined with the VITEK 2 Gram Positive Susceptibility Card-AST-GP68 (BioMerieux, Inc) using VITEK2 systems in the clinical laboratories of Rochester General Hospital. Results with 15 antibiotics were determined and the numerical values were expressed in ug/ml. The ability of the AST card to reliably detect resistance among S. pneumoniae to amoxicillin, linezolid and telithromycin is unknown. In our collection of S. pneumoniae, 17 isolates failed to give antibiotic susceptibility in the system.

Multilocus Sequence Typing (MLST)

Sequence types (STs) of S. pneumoniae isolates were determined using the MLST technique as described previously [12-16].

Statistics

To determine significant trends over time of otopathogen prevalence a variance-weighted least squares regression was used to model otopathogen colonization by the three years of interest (2008, 2009, and 2010). Similar tests were conducted to determine linear trends in oxacillin resistance and penicillin resistance in S. pneumoniae. To compare otopathogen prevalence rates from one year to the next, a chi-square test was conducted. For the aforementioned tests, a p-value of <0.05 was considered significant. All calculations were performed with STATA/SE 12.0 statistical software (College Station, TX, USA). In the analysis of NP colonization, if a child had the same S. pneumoniae isolate on sequential visits based on MLST then that data was included as a footnote in the Tables.

RESULTS

Study Population

Two hundred seventy-seven subjects were enrolled and had a study visit during the study period 1/1/2008 and 9/30/2010. There were a total of 1068 visits with 863 healthy well-child visits and 208 AOM visits. The majority of the subjects were male with a mean age of 7.9 months. Table 1 shows the demographic characteristics of the subjects.

Table 1.

Patient Demographics at Enrollment

Number of Patients	277
Sex (N, %)
Male	147 (53.1)
Female	130 (46.9)
Ethnicity/Race (N, %)
Non-Hispanic White	222 (80.1)
African American	38 (13.7)
Other	17 (6.1)
Age (months)
Mean	7.9
Range	4.1-38.1
Family History of OM (N, %)	131 (47.3)
Breastfeeding <6 months (N, %)	81 (29.2)
Exposed to Tobacco Smoke (N, %)	38 (13.7)
Daycare Attendance (N, %)	112 (40.4)

^*Missing data: 8 patients for daycare, 25 for breastfeeding status, and 7 for family History of OM, and 4 for smoking.

Table 2 shows the percentages of otopathogens isolated from the NP during well-child visits at 6, 9, 12, 15, 18, 24 or 30 months of age, at a time when the subjects were well, and from the NP and MEF at the time of an AOM episode. During healthy visits, S. pneumoniae colonization did not change significantly during the study time frame (no significant linear trend), including the PCV7 to PCV13 transition months during 2010. However, NTHi and M. catarhallis NP colonization during healthy visits significantly decreased from 2008 to October 2010 (p=0.048 and p=0.041 respectively). There was a higher NP colonization rate for all 3 otopathogens during an AOM episode compared with a time of health. The otopathogen distribution in the NP and causing AOM was stable during the study period 2008-October 2010. During an AOM episode, the otopathogen isolated from the MEF was also isolated from the NP, but 47% of the time along with other otopathogens (data not shown). S. pneumoniae colonization in the NP and MEF during episodes of AOM trended lower from 2008 to October 2010, but this decrease was not significant. NTHi and M. catarhallis NP colonization and MEF during episodes of AOM remained constant from 2008 to October 2010.

Table 2.

Otopathogen distribution causing colonization and AOM.

		Healthy	AOM
	Source	NP	NP	MEF
		N (%)
2008	Total visits	238	57	57
	Spn	73 (30.7)	34 (59.7)	19 (33.3)
	NTHi	36 (15.1)	25 (43.9)	16 (28.1)
	Mcat	106 (44.5)	29 (50.9)	10 (17.5)
2009	Total visits	276	78	78
	Spn	105 (38.0)	42 (53.8)	28 (35.9)
	NTHi	39 (14.1)	30 (38.5)	18 (23.1)
	Mcat	125 (45.3)	39 (50.0)	14 (17.9)
2010 (Jan. 1st-Sept. 30th)	Total visits	346	73	73
	Spn	112 (32.3)	33 (45.2)	19 (26.0)
	NTHi	34 (9.8)	36 (49.3)	25 (34.2)
	Mcat	127 (36.7)	36 (49.3)	5 (6.9)

Serotypes of S. pneumoniae

S. pneumoniae serotypes found during NP colonization at a healthy visit and at the time of an AOM episode as well as from the MEF are shown in Figure 1. Serotype 19A was the most commonly isolated serotype followed by serotype 15. There were no PCV7 serotypes isolated during the study period. The proportion of PCV13 serotypes isolated during NP colonization while healthy and during an AOM episode was 23%, 30% and 34% and 36%, 45% and 45% in 2008, 2009 and 2010, respectively. PCV13 serotypes were isolated from MEF in 47%, 52% and 58% of the AOM episodes in 2008, 2009 and 2010, respectively. No differences were detected in these observations during the PCV7 to PCV13 transition months compared with prior years.

Figure 1.

S. pneumoniae serotypes isolated during 2008-October 2010 in healthy colonized children, colonized AOM children and in the middle ear of children during AOM.

Antibiotic Susceptibility and β-lactamase production

In NP samples from healthy children, there was a significant increase in S. pneumoniae isolates that were oxacillin resistant from 2008-2010 (p =0.017). In NP and MEF samples at the time of AOM episodes, similar increasing trends in oxacillin resistant S. pneumoniae isolates were observed, but these trends were not significant as shown in Table 3.

Table 3.

Oxacillin resistant S. pneumoniae and β-lactamase producing NTHi distribution during health and AOM, 2008- October 2010.

			S. pneumoniae	NTHi
Children	Year	Source	Oxacillin Resistance	β-lactamase+
			N (%)
Healthy	2008	NP	14/73 (19.2)	9/36 (25.0)
	2009	NP	33/105 (31.4)	10/39 (25.6)
	2010 (Jan-Sept)	NP	38/105 (36.2)	6/34 (17.6)
AOM	2008	NP	13/34 (38.2)	12/25 (48.0)
		MEF	5/19 (26.3)	9/16 (56.2)
	2009	NP	18/42 (42.9)	5/30 (16.7)
		MEF	12/28 (42.8)	3/18 (16.7)
	2010 (Jan-Sept)	NP	17/33 (51.5)	10/36 (27.8)
		MEF	9/19(47.3)	8/25 (32.0)

Note: All the M. catarrhalis isolates were β-lactamase positive.

Significant difference was observed for oxacillin resistance Spn isolates during healthy vs. AOM NP colonization in 2008 only (p =0.03).

For NTHi NP samples during healthy visits from 2008-October 2010, there was an increase in β-lactamase producing isolates from 2008 to 2009, but then a decrease from 2009 to October 2010 (no significant trend). In NP and MEF samples at onset of AOM, β-lactamase producing NTHi isolates decreased from 2008 to 2009, but then increased from 2009 to October 2010 (no significant trend).

The antibiotic susceptibility of S. pneumoniae isolates from 2008 to October 2010 was tested against 15 antibiotics. For benzylpenicillin, there was an increasing trend for non-susceptible S. pneumoniae strains (intermediate + resistant) from 2008 to October 2010, but this trend was not significant (p =0.132). There was very little ceftriaxone resistance, 5.7%, 3.2% and 2.3% in 2008, 2009 and 2010 respectively. In 2008, there were 14 (26.4%) S. pneumoniae isolates that were resistant to 3 or more antibiotics, compared with 22 (17.6%) and 18 (20.9%) of S. pneumoniae isolates in 2009 and 2010 respectively.

Multi-locus Sequence Types

A total of 385 isolates of S. pneumoniae were characterized from 2008-October 2010 using MLST. One hundred twelve distinct sequence types (STs) were found, 47 of which had not been previously recorded. Several new alleles were also submitted to the MLST database; 2 aroe, 2 gki, 1 spi, 2 xpt and 1ddl. The most common sequence types (presented 4 or more times in the samples) and the serotypes associated with them for 2008, 2009 and 2010 are presented in SDC 1 (Table). In some cases different isolates with the same ST may express different capsules and where this was the case, the numbers of each serotype are shown in parenthesis. In each year, 2008, 2009 and 2010, the two most common STs found were 199 and 320. The ST 320 was usually associated with a 19A capsule, and ST 199 was associated with capsules 19A and 15. Since 19A and 15 were the most common capsular types found in the dataset from 2008-2010, a snapshot of all the STs belonging to capsular types 19A (see figure, SDC 2A) and 15 (see figure, SDC 2B) in three years was created using eBURST, displaying relationships between STs.

Comparisons were made between two groups of isolates; NP isolates that were found at the time of healthy asymptomatic NP colonization vs. NP isolates that were present at onset of AOM. SDC 3 (figure) shows the comparative eBURST diagram between isolates obtained from healthy colonized children and isolates that were found at onset of AOM during year 2008-October 2010. A CC6 contains STs that was found only during healthy colonization. CC6 belongs to capsular type 23 in our population from 2008-October 2010.

DISCUSSION

Knowledge of the otopathogens causing AOM, along with establishment of antibiotic susceptibility results, is required to guide empiric antibiotic selection. Tympanocentesis of MEF at onset of AOM provides the most reliable data for this needed information. PCVs containing 7, 10 and 13 serotypes are now in use in multiple countries. The composition of those vaccines was dictated by the known prevalent S. pneumoniae serotypes causing invasive and local respiratory infections. However, with the introduction of these vaccines emergence of replacement serotypes has occurred [2, 3, 9]. Thus, continuous monitoring is needed to guide future development of pneumococcal conjugate vaccines regarding which serotypes to add.

In this prospective study we found that S. pneumoniae has returned to be the most common otopathogen isolate in the time frame of 2008 through October 2010. As more time elapsed following the introduction of PCV7 the eliminated strains expressing capsular types in PCV7 have been increasingly replaced by other serotypes, especially serotypes 19A and 15. A significant proportion of the S. pneumoniae isolated in our study were penicillin non-susceptible, a trend we had previously noted in an earlier report [9]. Among the NTHi isolates from MEF, 16.7% to 56.2% produced beta lactamase, depending on the year of study. All the M. catarrhalis we isolated produced beta lactamase. This resistance mechanism renders the organisms non-susceptible to amoxicillin in regular or higher dosages.

We pursued a companion analysis of data generated from NP cultures. Prior reports have shown that NP cultures are not highly predictive of MEF isolates [17-26]. Our results re-confirmed that NP cultures at onset of AOM include the otopathogen causing the infection. However, because many children are colonized by multiple otopathogens at the same time, NP cultures cannot distinguish which among the otopathogens colonizing the NP actually causes AOM. Moreover, it would be more convenient to take NP cultures at times of health to track otopathogen distribution and antibiotic susceptibility, but we found that such cultures would be misleading compared with MEF results.

PCV13 was formulated to add serotypes of S. pneumonia that were not included in the PCV7 product and that had emerged as prevalent in the United States and Western Europe. Similarly, PCV10 was formulated with differing serotypes based on data from a different geography. Seven serotypes are common to PCV7 and PCV13 (4, 6B, 9V, 14, 18C, 19F, and 23F) and six additional serotypes are added in PCV13 (serotypes 1, 3, 5, 6A, 7F, and 19A). Early results from our center show that PCV13 may prove effective in preventing AOM caused by serotype 19A [27]. However, neither PCV10 nor PCV13 contain serotypes 15, 11, 23B and others we found to be prevalent in our study population. Our studies are ongoing and will continue to monitor the situation.

S. pneumoniae ST composition in 2008-October 2010 did not change significantly in our study population, similar to results from 2007-2011 among Massachusetts’s children [28, 29]. Like the study from Massachusetts, we found ST320 and 199 to be the most common in our population [29]. Because we had MEF isolates as well as NP isolates we also compared STs of strains isolated during NP colonization when children were healthy compared with NP colonization at onset of AOM; we found that STs generally did not differ in the two study cohorts. However, we did identify a small complex (CCL6) where all the strains were from asymptomatically colonized children suggesting that strains in that complex may be less virulent. All the strains in the CCL6 complex expressed capsular types 23A or 23B.

Penicillin non-susceptibility among S. pneumoniae and β-lactamase production by the gram-negative bacteria, NTHi and M. catarrhalis influence antibiotic selection. Higher dosages of amoxicillin improve eradication of penicillin non-susceptible S. pneumoniae, although MEF levels can be low in some children due to variable absorption of the drug [30]. Higher dosages of amoxicillin cannot overcome β-lactamase production by NTHi and M. catarrhalis. Our results suggest that high dose amoxicillin/clavulanate would be the best empiric choice for antibiotic treatment of AOM in preference to high dose amoxicillin. Antibiotics endorsed by the American Academy of Pediatrics as alternatives to amoxicillin/clavulanate [31] would also be appropriate.

Our study has limitations. Most of the children enrolled come from a single private pediatric practice in a single community; about 20% of the children in the study are referred to us from other community-based pediatric practices. Geographic variation may occur with regard to otopathogen distribution and antibiotic susceptibility, although a recent comparison of results from our center and Pittsburgh PA found a high degree of similarity among centers [32].

In summary, continued reappraisal of the bacteria causing AOM is vital to the public health because the situation is continuously changing under the influence of antibiotic use and vaccination. Tympanocentesis-identified middle ear isolation of otopathogens is the gold standard for accomplishing that goal. We are now studying the efficacy PCV13in preventing AOM and modifying NP colonization by otopathogens, commencing from October 2010.