Moraxella catarrhalis is a prominent pathogen that causes acute otitis media in children and lower respiratory tract infections in adults (such as exacerbations of chronic obstructive pulmonary disease) (Hol et al., 1996; Ahmad, 1998), resulting in significant socio-economic burden on healthcare systems globally.

Previous studies, including our own (Wang et al., 2011; Flamm et al., 2012; Liu et al., 2012, 2015; Tang et al., 2016), have reported on the increased prevalence of macrolide-resistant M. catarrhalis in Mainland China, ranging from 40 to 80% in children, and 5–10% in adults. A better understanding of the evolutionary path of the organism from macrolide-susceptible to macrolide-resistant M. catarrhalis, is crucial for controlling the spread of macrolide resistance. However, based on our previous studies, showing 40–60% macrolide resistance amongst M. catarrhalis isolates from healthy toddlers (12–18 months) (Liu et al., 2012), and that most macrolide resistant isolates (5/7) were obtained from patients who had not received macrolide antibiotics within the previous 30 days (Liu et al., 2015), we think that macrolide resistance is acquired.

Multilocus sequence typing (MLST) has been widely employed in epidemiological investigations of various scales (Cox et al., 1995; Enright and Spratt, 1998; Maiden et al., 1998; Enright et al., 2001; Qin et al., 2009), including population, pathogenicity, and evolution studies, of several bacteria. To gain insights into the molecular evolution of macrolide resistance in M. catarrhalis, PCR and sequence analysis of the 23S rRNA gene, copB typing, MLST, and macrolide susceptibility testing, were performed on 181 M. catarrhalis isolates.

CopB is one of the several M. catarrhalis outer membrane proteins that elicit a systemic humoral immune response in humans, resulting in the production of serum antibody specific for CopB, which underscores the potential of CopB as a vaccine candidate (Meier et al., 2003; Murphy et al., 2005; Liu et al., 2006). The copB gene is relatively conserved in M. catarrhalis, and has been detected in all isolates examined so far (Liu et al., 2012, 2015). Digestion of the copB products with RsaI restriction enzyme divides the organism into four copB types, namely copB type 0 (374 and 157 bp), type I/III (342 and157 bp), type II (332 and 187 bp) and type IV (519 bp) (Verhaegh et al., 2008), and has already been used in several typing studies (Verhaegh et al., 2008; Liu et al., 2012, 2015).

In our previous studies, we found that there was a trend toward an increase in copB types I/III, and a decrease in copB type II, in adult associated M. catarrhalis isolates, when compared with isolates from children (Liu et al., 2012, 2015). The present findings are somewhat in agreement to these previous results although the difference was not statistically significant, which suggests that the copB type may not be age-related.

When the allelic profiles of MLST were analyzed using Bionumerics software, population diversity was clearly demonstrated (Figure 2). However, four likely primary founders (ST224, ST363, STN08, and STN10) were demonstrated in CCs CC224, CC363, CCN08, CCN10. The predicted primary founder ST224, with the largest number and complicated links to other STs, is likely to be the ancestor of all macrolide- susceptible isolates. And all the five clusters encompassing macrolide-resistant M. catarrhalis isolates, located at the edge of the population snapshot (Figure 2) with less links to CC224, seem to have evolved in several ways from macrolide-susceptible isolates, but not exactly only from CC224.

One important finding of this study is the description of two macrolide-resistant CCs, CCN10 and CC363, and the likely primary founders STN10 and ST363. As illustrated in Figure 2, the two valuable macrolide-resistant CCs, CCN10 and CC363, are significantly separated from other clusters including CC224, and from the year collected. STN10 and ST224 were first isolated in 2010 and 2012, respectively, from which we surmise that CCN10 is less likely to have evolved from CC224. The small number of isolates analyzed over a short duration (2010–2014) makes it difficult to come up with firm conclusions. Some valuable insights might be gained from studying a large number of isolates collected over a 10 or 12 years period.

A limitation of this study is that the isolates originated from two different hospitals; the prevalence and distribution of macrolide-resistance or STs might be totally different in different areas. Actually, antimicrobial susceptibility testing and MLST typing were first performed on isolates from PUMCH adults, and seven macrolide-resistant isolates and some important STs such as ST224, STN10, and STN16, were detected. This encouraged us to examine whether the macrolide-resistant population was concentrated in selected STs. PUMCH is located in the northern part of China and the patients are mainly adults, while FAHXU is situated in the southern part, and is well known for pediatric infectious diseases. So the two hospitals were ideal locations for our study purpose, including comparing the distribution of different STs and macrolide-resistant isolates in the two hospitals (Figure 2; Supplementary Figure S1). From the results we can see that even though the distribution of different STs is definitely related to regions, some important STs such as ST224, STN10, and STN16, were detected in both hospitals. Moreover, most STs of macrolide-resistant isolates (except for STN10 and STN16) detected in PUMCH or FAHXU belonged to the same clusters (such as both STN01 and ST331 belonged to cluster 6) (Figure 2; Supplementary Figure S1), which suggests that these STs might be very critical clones in the process of evolution, especially on macrolide resistance. And we can also see that macrolide resistance is mainly age-related without obvious connection with different regions (Figure 2; Supplementary Figures S1 and S2), albeit limited data from the two hospitals. However, even when the results from adults and children in the same hospital were analyzed (Supplementary Figure S3), most of the derived conclusions are somehow confirmed.

Further study limitations include the relatively small number of macrolide-resistant M. catarrhalis isolates in adults compared with macrolide- susceptible isolates, which may influence the reliability of results to some extent. In addition, STN01–STN31 have not been submitted to http://mlst.warwick.ac.uk/mlst/dbs/Mcatarrhalis/, so these ST names were defined in the present study. Moreover, we compared isolates collected over different time periods in the two hospitals; strains from PUMCH were collected from 2010 to 2013 while the ones from FAHXU were collected in 2014. In our further study, the genomic sequences of these strains would be determined according to the requirements of the Enterobase¹ instead of the MLST website, and then the accurate STs would be given.

Y-LL, MX, TK, and FK wrote the manuscript. J-WC, Z-PX, and H-PX collaborated in molecular investigations of the strains. SY and W-JZ summarized the patient’s medical records. Y-CX designed and supervised the study.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.