Ampicillin resistance of invasive Haemophilus influenzae isolates in Germany 2009–2012

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Ampicillin resistance of invasive Haemophilus influenzae isolates in Germany 2009–2012 Thiên-Trí Lâm ∗ , Heike Claus, Johannes Elias, Matthias Frosch, Ulrich Vogel Institute for Hygiene and Microbiology, National Reference Centre for Meningococci and Haemophilus influenzae, University of Würzburg, Josef-Schneider-Str. 2 (E1), 97080 Würzburg, Germany

a r t i c l e

i n f o

Keywords: Epidemiology Invasive infection Laboratory surveillance ftsI Beta-lactamase BLNAR

a b s t r a c t In this retrospective study covering a four-year observation period (2009–2012) the prevalence of aminopenicillin resistance of invasive Haemophilus influenzae (Hi) in Germany was analyzed. The main resistance mechanism against aminopenicillins is conferred by ␤-lactamase production, which can be inhibited by clavulanate or sulbactam. Apart from that, ␤-lactamase negative ampicillin resistance (BLNAR) has been reported due to mutations in the penicillin-binding protein PBP3. The prevalence of BLNAR varies considerably in different countries. Representative data from Germany have not been reported. We analyzed 704 culture positive cases with bacteraemia or detection of Hi in cerebrospinal fluid; 82 isolates (11.6%) were phenotypically resistant to ampicillin. Among these isolates, 65 (79.3%) showed ␤-lactamase production, and 17 isolates (20.7%) were phenotypic BLNAR Hi. The proportion of ampicillin resistant isolates remained stable over the observation period. Analysis of the PBP3 sequences of 133 isolates with different susceptibility phenotypes including susceptible, BLNAR, and ␤-lactamase positive isolates, revealed a high genetic diversity. Previously described PBP3 mutations were associated to elevated MIC values, albeit not exclusively, since few highly susceptible strains were found to be positive for the mutations. Furthermore, since ampicillin susceptible strains with elevated MIC values frequently harboured these mutations, prediction of the resistance phenotype using ftsI sequencing appears to be impossible. © 2015 Elsevier GmbH. All rights reserved.

1. Introduction Meningitis and septicaemia are the most important invasive infections due to the gram-negative bacillus Haemophilus influenzae (Hi). Aminopenicillins and cephalosporins are the drugs of choice for the treatment of Hi infections. To date, resistance of Hi to 3rd generation cephalosporins has been described only infrequently (Garcia-Cobos et al., 2007; Leclercq et al., 2013). However, resistance against aminopenicillins alone or in combination with ␤lactamase inhibitors can be found in varying percentages ranging from 2% up to >60% (Fluit et al., 2005; Giufre et al., 2011; Hoshino et al., 2013; Jansen et al., 2008; Ladhani et al., 2008; Park et al., 2013; Puig et al., 2014; Resman et al., 2012; Setchanova et al., 2013; Sill and Tsang, 2008; Skaare et al., 2010; Ubukata et al., 2013; Witherden et al., 2011). Two basic mechanisms leading to reduced susceptibility to aminopenicillins have been described (see review in Needham,

∗ Corresponding author. E-mail address: [email protected] (T.-T. Lâm).

1988; Tristram et al., 2007). Production of ␤-lactamases leads to ampicillin resistance (BLPAR) that can be reversed by ␤-lactamase inhibitors and is usually associated with high ampicillin minimal inhibitory concentrations (MIC). Thereby TEM-1 ␤-lactamase is predominantly found in Hi isolates (Sykes et al., 1975; Williams et al., 1974). ROB-1, the second ␤-lactamase type known in Hi is only found in very few cases (Rubin et al., 1981). Reduced susceptibility to aminopenicillins can also be caused by mutations of the penicillin binding protein PBP3 (Clairoux et al., 1992; Mendelman et al., 1990; Parr and Bryan, 1984), a protein involved in cell division that is encoded by ftsI (Malouin et al., 1990; Weiss et al., 1997). Isolates exhibiting this resistance type are named ␤-lactamase negative ampicillin resistant (BLNAR) Hi and show lower ampicillin MIC-values than their BLPAR counterparts. In addition, reduced aminopenicillin susceptibility cannot be reversed by ␤-lactamase inhibitors. Combination of both resistance mechanisms leads to organisms that display ␤-lactamase production that cannot be inhibited by clavulanate, so-called ␤-lactamase positive amoxicillin–clavulanate resistant (BLPACR) Hi. A variety of amino acid (AA) substitutions in PBP3 compared to the sequence of type strain RD KW20 have been described by

http://dx.doi.org/10.1016/j.ijmm.2015.08.028 1438-4221/© 2015 Elsevier GmbH. All rights reserved.

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different authors. Ubukata et al. (2001) came up with a classification of BLNAR by using an in vitro penicillin binding assay to categorize the resistance phenotype of clinical isolates according to the PBP3 mutations. They identified three groups of AA substitutions leading to reduced ampicillin susceptibility. R517H, near the conserved KTG-motif, is defined for group I and N526K for group II. In group III, in addition to the N526K mutation, M377I, S385T, and L389F are found near the conserved SSN motif. Although several authors have proposed subdivisions of this classification (Dabernat et al., 2002; Garcia-Cobos et al., 2007; Hasegawa et al., 2006), Hotomi et al. (2007) describe strains negative for the ␤-lactamase (bla) gene as genetically ␤-lactamase-negative ampicillin-resistant (gBLNAR). Furthermore, data indicating that changes in PBP3 often result in reduced ampicillin susceptibility on a low level (Hasegawa et al., 2006; Sanbongi et al., 2006) led to the term low BLNAR (Tristram et al., 2007). The confusing complexity in the nomenclature of BLNAR reflects the enormous diversity of PBP3 mutations, which to date is not fully understood. The geographic distribution of BLNAR seems to vary substantially with highest prevalence in Japan (Hasegawa et al., 2006; Hoshino et al., 2013), whereas BLNAR are relatively rare in Europe (Fluit et al., 2005; Jansen et al., 2008) and the USA (Hasegawa et al., 2003). In many reports, Hi strains from airway infections have been analyzed. The aim of this study was to contribute to the surveillance of antibiotic resistance in Hi isolates recovered from bloodstream or cerebrospinal fluid infection. We present the first prevalence data for ampicillin susceptibility in invasive Hi isolates collected in Germany from 2009 to 2012 with a genotypic characterization of BLNAR and BLPACR. 2. Methods 2.1. Clinical isolates As part of the German laboratory surveillance system accompanying the statutory notification in Germany, Hi strains from bloodstream or CSF infections were submitted to the National Reference Laboratory for Meningococci and H. influenzae (NRZMHi). Species diagnosis was confirmed phenotypically by positive oxidase reaction and factor-dependent growth on BBL Hemo ID QUAD plates (BD, Heidelberg, Germany). The presence of the H. influenzaespecific ompP2 gene (Hobson et al., 1995) was confirmed by PCR. All isolates were serotyped by slide agglutination combined with detection of bexA and serotype-specific genes (Falla et al., 1994; Lam et al., 2011). 2.2. Antibiotic resistance analysis Minimal inhibitory concentrations (MIC) of antibiotics were determined using gradient agar diffusion tests (Etest, bioMérieux, Nürtingen, Germany). Inoculum suspensions were prepared in brain heart infusion medium (BHI, Becton Dickinson, Heidelberg, Germany) and adjusted to a McFarland 0.5 turbidity standard using a DensiCHEKTM Plus instrument (bioMérieux, Nürtingen, Germany). The inocula were plated on Mueller-Hinton agar with 5% defibrinated horse blood and 20 mg/L ␤-NAD (MH-F, Oxoid Deutschland GmbH, Wesel, Germany). Breakpoints were interpreted according to the guidelines of the European Committee for Antibiotic Susceptibility Testing (EUCAST, 2014). H. influenzae ATCC 49247 was used to for quality control of the test media and Etest strips. Nitrocefin tests (Fluka/Sigma Aldrich, St. Louis, MO, USA) were performed on all isolates to detect ␤-lactamase expression. Beta-lactamases were subsequently characterized by PCR for TEM1 and ROB-1 (Hasegawa et al., 2003). Isolates with ampicillin

MIC > 1 mg/L, but without ␤-lactamase were defined as BLNAR. Hi with an ampicillin MIC > 1 mg/L and ␤-lactamase expression were further tested for resistance against amoxicillin–clavulanate. Resistant isolates were defined as BLPACR. Ampicillin resistant isolates (MIC > 1 mg/L) without reduced susceptibility to amoxicillin–clavulanate were defined as BLPAR. Beta-lactamase positive ampicillin resistant isolates with an amoxicillin–clavulanate MIC > 2 mg/L were defined as BLPACR. Sequencing of the ftsI gene in BLNAR and BLPACR Hi isolates and comparison of the encoded PBP3 sequence with the reference sequence of strain RD KW20 was carried out as has been described elsewhere (Ubukata et al., 2001). Thereby, the relevant section starting at AA330 before the highly conserved motif STVK up to AA530 located behind the conserved KTG motif was analyzed. Likewise, PBP3 sequences of ␤-lactamase positive strains and isolates with ampicillin MIC = 0.016 to 1 mg/L were examined. 2.3. Data processing For sequence analysis of PBP3, forward and reverse ftsI sequences were compiled, translated and aligned using the DNASTAR Lasergene suite version 10.0.1 (DNASTAR, Madison, WI, USA). Isolate data were extracted from the NRZMHi data base and quantitatively analyzed using Microsoft Excel 2013, version 15.0 (Microsoft, Redmond, WA, USA). Statistical analysis, where necessary, was performed using Pearson’s Chi Square Test, and statistical significance was assumed with p < 0.05. 3. Results 3.1. Epidemiology and isolate characteristics The NRZMHi received isolates from 147 different diagnostic laboratories. Submissions from a total of 710 patients with Hi bloodstream or CSF infections were processed during the four year observation period from 2009 to 2012. The incidence rate, as registered by the national statutory notification system was 0.23, 0.26, 0.33, and 0.40 per 100,000 inhabitants for 2009, 2010, 2011, and 2012, respectively (Robert Koch Institut, Survstat@RKI 2.0, https:// survstat.rki.de, data accessed March 16th 2015). 706 vital isolates could be serotyped and tested for antibiotic resistance. All isolates derived from individual patients, the male to female ratio was 51 to 49. The patient age ranged from 1 day up to 100 years, the median age was 69. The majority of isolates lacked the capsule transport gene bexA and was non-typeable (NTHi; n = 549; 77.8%), and 157 isolates were capsulated (22.2%). Among the capsulated strains, 36 (22.9%) were serotype b (Hib). No so-called Hib− strains were found. A detailed break-down of the laboratory surveillance data including phenotypic and genotypic capsule serotype analysis is subject of a separate study (Lam et al., manuscript in preparation). 3.2. Distribution of ampicillin resistance Ampicillin resistance according to EUCAST (resistance breakpoint >1 mg/L) was found in 82 isolates (11.6%), whereas the majority of isolates (n = 624; 88.4%) was ampicillin susceptible. The proportion of ampicillin resistant isolates remained at a similar level throughout the observation period (p > 0.05; Fig. 1). The mean age for patients with ampicillin resistant and ampicillin susceptible isolates did not differ significantly. Isolates from patients aged ≥60 years (n = 458) showed the same proportion of ampicillin resistance as strains from cases <60 years (n = 248; p > 0.05). Sixty-five isolates (79.3% of resistant isolates) showed ␤lactamase production. Genetic analysis was positive for TEM-1 ␤-lactamase in all of these cases, whereas ROB-1 ␤-lactamase was not detected. Of all 65 ␤-lactamase producing isolates, six (9.2%)

Please cite this article in press as: Lâm, T.-T., et al., Ampicillin resistance of invasive Haemophilus influenzae isolates in Germany 2009–2012. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2015.08.028

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Fig. 1. Prevalence of ampicillin resistance phenotypes 2009–2012. The numbers of ␤-lactamase negative ampicillin susceptible (BLNAS; ampicillin MIC ≤ 1 mg/L), ␤-lactamase positive ampicillin resistant (BLPAR; ampicillin MIC > 1 mg/L), and ␤-lactamase negative ampicillin resistant (BLNAR) isolates are indicated within the columns. In BLPAR is the number of ␤-lactamase positive amoxicillin–clavulanate resistant (BLPACR; amoxicillin–clavulanate MIC > 2 mg/L) included (2009: 1; 2010: 1; 2011: 2; 2012: 2).

were resistant to amoxicillin–clavulanate (MIC > 2 mg/L) and were consequently classified as BLPACR. All of these isolates showed high ampicillin MICs ranging from 24 mg/L (one isolate) and 32 mg/L (one isolate) up to ≥256 mg/L (the remaining four isolates). In 17 isolates (20.7% of resistant isolates), no ␤-lactamase was detected in spite of phenotypic ampicillin resistance (BLNAR). The ampicillin MIC of 14 BLNAR isolates was 1.5 mg/L, and the MIC of the three remaining isolates did not exceed 4 mg/L. 3.3. Genetic analysis of ampicillin resistance types Sequencing of the ftsI gene from a total of 157 isolates was carried out to detect mutations in the PBP3 sequence from L338 up to N530. This number includes 17 BLNAR, six BLPACR and 59 BLPAR isolates found by phenotypic resistance testing. Since there is no consensus on the definition of BLNAR and because different authors use different MIC breakpoints for the interpretation of ampicillin susceptibility, 16 additional isolates with an ampicillin MIC of 1 mg/L were examined for PBP3 mutations. The ftsI of 35 ␤lactamase negative isolates with ampicillin MICs 0.5 or 0.75 mg/L was sequenced to further examine the occurrence of low BLNAR isolates. In addition, PBP3 was sequenced of 24 isolates with lowest ampicillin MIC ranging from 0.016 to 0.125 mg/L as controls. An overview of the results is given in Table 1. The most frequent AA substitutions found were N526K (n = 73; 46.5% of 157 PBP3 sequences analyzed), D350N (n = 69; 43.9%) and M337I (n = 52; 33.1%). Moreover, A502 was substituted 60 times, 43 times by valine, seven times by threonine, and once by serine. The mutation N526K is defining group II BLNAR and co-occurred frequently, but not always, with D350N as has been described before (Ubukata et al., 2001). In contrast to the study of Ubukata et al. (2001), the replacement of serine at position 357 by asparagine has not been found in isolates with N526K.

Table 1 Summary of the PBP3 mutations found in different resistant phenotypes. The number of isolates of a PBP3 mutation pattern is grouped according to the phenotypic ampicillin resistance found [PBP3 mutational group according to Ubukata et al. (2001)]. Ampicillin susceptible isolates with relevant PBP3 mutations are considered genotypic BLNAR (gBLNAR) according to Hotomi et al. (2007). PBP3 mutational groupa Phenotype

n

Wild type

No group

I

All AMP 0.016–0.125 AMP 0.5 AMP 0.75–1 BLNAR BLPAR BLPACR

157 24 10 41 17 59 5

18 1 2 0 47 0

3 1 1 0 5 1

1b 0 1b 0 0 0

II

III

2b 8b 37b 15 7 4

0 0 0 2 0 0

Abbreviations: AMP—ampicillin; BLNAR—␤-lactamase negative ampicillin resistant; BLPAR—␤-lactamase positive ampicillin resistant; BLPACR—␤-lactamase positive amoxicillin–clavulanate resistant; MICs are indicated in mg/L. Group I: R517H. Group II: N526K. Group III: M377I, S385T, L389F. a According to Ubukata et al. (2001). b gBLNAR.

Occurrence of the most frequent AA substitution N526K was also found in two Hi isolates with the very low ampicillin MICs of 0.047 and 0.094 mg/L. One additional isolate with ampicillin MIC 0.016 mg/L showed mutation R517H together with A502T, which defines group I BLNAR isolates according to (Ubukata et al., 2001). Out of 35 isolates with ampicillin MIC of 0.5 or 0.75 mg/L, 30 turned out to be group II showing the characteristic N526K substitution, often together with other mutations (17 with A502 V and at least one of the mutations D350N or G490E; 3 with A502T; 3 I449V; according to Dabernat et al. (2002) corresponding to subdivisions IIb, IIc, IId, respectively). Two isolates did not show any mutation in the PBP3 section examined.

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Among the 17 phenotypic BLNAR isolates fifteen had the AA substitution N526K qualifying them as group II BLNAR (Ubukata et al., 2001), usually combined with further mutations (according to Dabernat et al., 2002 eleven group IIb and two group IIc). Two isolates showed a mutation pattern fitting to group III (M377I, S385T, L389F) with the additional mutations D350N, S357N, R517H and T532S, which has been called group III-like (Garcia-Cobos et al., 2007). In spite of the numerous mutations, the one of these isolates showed an ampicillin MIC of 1.5 mg/L. In the other III-like BLNAR isolate the ampicillin MIC of 4 mg/L was the highest of all BLNAR isolates tested in this study, but still moderate relative to the ampicillin MIC found in ␤-lactamase producing Hi. Four of the BLPACR isolates, among them two with ampicillin MIC ≥ 256 mg/L, showed mutations of group II (three of group IIb and one with mutations of both group IIb and IId). The BLPACR Hi with highest ampicillin as well as amoxicillin–clavulanate MICs showed only D350N. An overview of the mutations found in phenotypic BLNAR and BLPACR is given in Table 2. In sum, we found mutations that have been published to define BLNAR including the very frequent mutation N526K to be present in all 17 BLNAR Hi strains, whereas only 3 of 24 strains with lowest ampicillin MIC harboured these mutations. The finding suggests an association of the mutations with BLNAR, however, it is unlikely that they exclusively determine resistance. Many strains with MIC of 0.5 to 0.75 mg/L, which are not considered resistant according to EUCAST, also harboured the mutations (Fig. 2). This observation suggests that the mutations contribute to enhanced MICs, but that on the other hand they cannot be used to genetically predict the resistance phenotype.

4. Discussion In this study, ampicillin resistance data were analyzed for 706 Hi isolates from blood and cerebrospinal fluid. These clinical strains were collected 2009 to 2012 by the NRZMHi from nationwide submissions. The proportion of about 10% ampicillin resistant invasive isolates has remained stable over the four year observation period. A statistically not significant peak of almost 15% was observed in 2011. There is currently no consensus on how to define BLNAR Hi, because different ampicillin resistance breakpoints are used and because it is unclear whether gBLNAR are clinically valid. In our study, we used restrictive criteria focusing on isolates from cerebrospinal fluid and blood, and BLNAR isolates were defined in the first place by their phenotypic ampicillin MIC in vitro on the basis of EUCAST breakpoints. This may have led to lower prevalence rates than in other studies. Few antibiotic resistance data have been published from national surveillance programmes on invasive H. influenzae disease. Here, we present the first Hi aminopenicillin susceptibility data from bloodstream and CSF isolates as part of the German National Laboratory Surveillance that complements statutory notification. Many reports on the antibiotic susceptibility of Hi in different European countries focus on respiratory tract infections or do not define the disease isolates (Barbosa et al., 2011; Dabernat et al., 2002; Fluit et al., 2005; Jansen et al., 2008; Perez-Trallero et al., 2010; Skaare et al., 2010). Recent national reports with detailed analysis of antibiotic resistance in invasive Hi infections exist for a number of European countries. In a study that included invasive as well as respiratory tract infections, Setchanova et al. (2013) reported an ampicillin resistance rate of 22% among the cohort of invasive isolates from Bulgaria. Some countries reported an increase in the prevalence of BLNAR (Giufre et al., 2011; Resman et al., 2012). During the four years of observation in Germany, an increase of the proportion of BLNAR was not observed.

A thorough overview of antibiotic susceptibility in H. influenzae isolates from blood and CSF has been published for England and Wales (Ladhani et al., 2008). The ampicillin resistance rate of 16.2% was acquired from the laboratory surveillance programme of the Health Protection Agency and was defined using the British Society for Antimicrobial Chemotherapy criteria. The constantly low prevalence of BLNAR isolates found in England and Wales is in line with the ampicillin resistance observed over the years in Germany. However, a more detailed genotypic analysis of BLNAR isolates was not carried out in that study. In a very recent publication, isolates from various primarily sterile sites from an active national laboratory surveillance programme were analyzed for antibiotic resistance (Garcia-Cobos et al., 2014). The prevalences of 19.5% and 1.2% for ampicillin resistance and phenotypic BLNAR isolates according to EUCAST interpretation criteria reflect the proportions we have found in our study (11.6% and 2.4%, respectively). Moreover, consistent with our own findings are the frequent occurrences and a high diversity of mutations in the PBP3 sequence in the Hi isolates tested in that study. The ampicillin resistance rate we found in Hi from blood and CSF is in line with previous reports on blood isolates (Hoban et al., 2001) and supports treatment guidelines, that do not recommend aminopenicillins, but third generation cephalosporins as first line therapy for invasive Hi infections (Tunkel et al., 2004). Further studies are needed to elucidate the susceptibility of invasive Hi for cephalosporins in Germany. This is of particular interest, since the incidence of invasive Hi infections caused by unencapsulated Hi, however at low levels, has been increasing in Germany as reported for other European countries (Ladhani et al., 2010). Our results indicate that only a few substitutions are linked to higher ampicillin MICs. These substitutions mostly belong to group III and group III-like. This is in line with other studies characterizing these mutations as “high resistance genotypes”, whereas the group II mutation has been considered a “low resistance genotype” (Skaare et al., 2014). As our results and observations from other authors show, isolates bearing group I or group III mutations are extremely rare in invasive disease, whereas the N526K mutation, which is characteristic to group II, can be found frequently and even occurs in isolates with very low ampicillin MIC. The N526K mutation has in fact been taken by some authors as a hallmark to define BLNAR Hi genetically coining the term gBLNAR (Hotomi et al., 2007), and different authors have undertaken the effort to find phenotypic screening tests to detect isolates bearing the N526K mutation (Garcia-Cobos et al., 2008; Norskov-Lauritsen et al., 2011). Genetic studies using site directed mutagenesis and genetic recombinants have shown the impact of known PBP3 mutations on the susceptibility to aminopenicillins and cephalosporins in vitro: Osaki et al. (2005) found an increase of the ampicillin MIC from 0.125 mg/L (wild type) to 0.25 mg/L (recombinant) upon introduction of the mutation N526K. However, the role of the mutations for the antibiotic resistance in vivo and its consequences for the treatment of the patient remain obscure. Animal studies are needed to determine the in vivo activity of aminopenicillins during treatment of invasive Hi infections in correlation to in vitro MICs. The current evidence supporting mutation analysis as the major guidance for resistance interpretation is not sufficient. Based on our results, the mutation N526K is too frequently found to predict relevant ampicillin resistance. In our study, it was even present in isolates with an ampicillin MIC as low as 0.047 and 0.094 mg/L. Interestingly, the two BLPACR isolates with highest ampicillin as well as amoxicillin–clavulanate MICs did not show any relevant PBP3. Therefore, the analysis of gBLNAR seems not practical, since a mutation or a mutation pattern is not unique to a specific resistance phenotype, and since it is the phenotype that ultimately leads to clinical consequences.

Please cite this article in press as: Lâm, T.-T., et al., Ampicillin resistance of invasive Haemophilus influenzae isolates in Germany 2009–2012. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2015.08.028

AMP

− − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − − + + + + +

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 2 3 4 24 32 256 256 256

AMC

Phenotype

3 3 3 4 5

BLNAS BLNAS BLNAS BLNAS BLNAS BLNAS BLNAS BLNAS BLNAS BLNAS BLNAS BLNAS BLNAS BLNAS BLNAS BLNAS BLNAR BLNAR BLNAR BLNAR BLNAR BLNAR BLNAR BLNAR BLNAR BLNAR BLNAR BLNAR BLNAR BLNAR BLNAR BLNAR BLNAR BLPACR BLPACR BLPACR BLPACR BLPACR

L338

D350

S357

V362

A368

M377

S385

L389

A437

T443

I449

G490

K494

A502

N350 N350 N350 N350 N350

I377 I377 I377 I377 I377

N350

I377

V502

N350

I377

V502

E490

V509

R517

V502 V502 V502 V502 V502 V502

K526 K526 K526 K526 K526 K526 K526 K526

E490

K526 K526 K526 K526 K526 K526 K526 K526

V449 N350 N350 N350 N350

E490 E490 E490

I377 I377 I377 T368

N350

N357

I377

T385

H517 E490

I338

N350 N350 N350 N350 N350 N350 N350 N350 N350 N350 N350 N350 N350 N350 N350 N350 N350 N350 N350

I377 I377 I377

N357

T368

I377 I377 I377 I377 I377 I377 I377 I377 I377 I377 I377 I377

E490

E490

E490 T385

V502 V502 V502 T502

F389

F389

E490 I377

N526

V449

V502 V502 V502 V502 T502 V502 V502 V502 V502 V502 V502 V502 V502 V502 V502 V502 V502 V502 V502

K526 K526 K526 K526 K526 K526 K526 K526 K526 K526 K526 K526 K526 K526 H517 K526 K526 K526 K526

A530

S530

S530

S530

Group IIb IIb IIb IIb IIb II IIa IIb WT IIa IIb IId IIa IIb IIb IIb IIc III-like IIb IIb IIb IIb IIc IIb IIb IIb IIb IIb IIb IIb IIb IIb III-like IIb IIb IIb IIb,IId no

Abbreviations: AMP—ampicillin; Lac—␤-lactamase; AMC—amoxicillin–clavulanate. MICs are indicated in mg/L, WT—wild type.

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Lac

H613 H727 H791 H823 H843 H844 H891 H896 H959 H994 H1082 H1122 H1139 H1155 H1209 H1244 H500 H705 H795 H807 H850 H918 H941 H979 H1021 H1099 H1101 H1110 H1224 H1236 H1090 H1182 H1257 H670 H491 H839 H950 H1270

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Table 2 Amino acid substitutions found in the PBP3 sequence of invasive ␤-lactamase negative ampicillin resistant (BLNAR) and ␤-lactamase positive amoxicillin–clavulanate resistant (BLPACR) H. influenzae isolates. Included are also ␤-lactamase negative ampicillin susceptible (BLNAS) isolates with a borderline ampicillin MIC of 1 mg/L. Individual isolates are identified by the strain collection number. The PBP3 mutation pattern is grouped according to Ubukata et al. (2001).

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Fig. 2. Phylogenetic tree based on the PBP3 sequences from AA330-530 of all ␤-lactamase negative isolates analyzed in this study. Resistance phenotypes are indicated next to the isolate number: AMP—ampicillin (MIC indicated in mg/L). The PBP3 of reference strain RD KW20 was defined as wild type (WT). PBP3 mutations in comparison to this reference are indicated for every group.

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Please cite this article in press as: Lâm, T.-T., et al., Ampicillin resistance of invasive Haemophilus influenzae isolates in Germany 2009–2012. Int. J. Med. Microbiol. (2015), http://dx.doi.org/10.1016/j.ijmm.2015.08.028