MICs Of Β-lactams Research Articles

The Spx stress regulator confers high-level β-lactam resistance and decreases susceptibility to last-line antibiotics in methicillin-resistant Staphylococcus aureus.

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a leading cause of mortality worldwide. MRSA has acquired resistance to next-generation β-lactam antibiotics through the horizontal acquisition of the mecA resistance gene. Development of high resistance is, however, often associated with additional mutations in a set of chromosomal core genes, known as potentiators, which, through poorly described mechanisms, enhance resistance. The yjbH gene was recently identified as a hot spot for adaptive mutations during severe infections. Here, we show that inactivation of yjbH increased β-lactam MICs up to 16-fold and transformed MRSA cells with low levels of resistance to being homogenously highly resistant to β-lactams. The yjbH gene encodes an adaptor protein that targets the transcriptional stress regulator Spx for degradation by the ClpXP protease. Using CRISPR interference (CRISPRi) to knock down spx transcription, we unambiguously linked hyper-resistance to the accumulation of Spx. Spx was previously proposed to be essential; however, our data suggest that Spx is dispensable for growth at 37°C but becomes essential in the presence of antibiotics with various targets. On the other hand, high Spx levels bypassed the role of PBP4 in β-lactam resistance and broadly decreased MRSA susceptibility to compounds targeting the cell wall or the cell membrane, including vancomycin, daptomycin, and nisin. Strikingly, Spx potentiated resistance independently of its redox-sensing switch. Collectively, our study identifies a general stress pathway that, in addition to promoting the development of high-level, broad-spectrum β-lactam resistance, also decreases MRSA susceptibility to critical antibiotics of last resort.

In vitro activity of cefiderocol against Pseudomonas aeruginosa demonstrating evolved resistance to novel β-lactam/β-lactamase inhibitors.

Cefiderocol demonstrates excellent activity against MDR Pseudomonas aeruginosa; however, the activity against isolates from patients previously treated with β-lactam agents is unknown. We aimed to determine the activity of cefiderocol against P. aeruginosa collected before and after treatment with traditional β-lactams and new β-lactam/β-lactamase inhibitors. Cefiderocol MICs were determined in triplicate in iron-depleted cation-adjusted Mueller-Hinton broth and compared with β-lactam MICs tested by standard methods. All isolates underwent WGS analysis to identify mutations associated with resistance. One hundred and seventy-eight P. aeruginosa isolates were evaluated; 48% (86/178) were non-susceptible to ceftazidime/avibactam, ceftolozane/tazobactam and/or imipenem/relebactam. The cefiderocol MIC50 and MIC90 were 0.12 and 1 mg/L, respectively. Median cefiderocol MICs did not vary against isolates classified as MDR, XDR, or those non-susceptible to ceftazidime/avibactam, ceftolozane/tazobactam and/or imipenem/relebactam when compared with non-MDR isolates. Against isolates collected from patients previously treated with ceftolozane/tazobactam, cefiderocol MICs were increased 4-fold compared with baseline. Cross-resistance to cefiderocol was identified in 21% (3/14) of patients who developed treatment-emergent resistance to ceftolozane/tazobactam. Overall, 6% (11/178) of isolates demonstrated cefiderocol MICs ≥2 mg/L, which were disproportionately collected from patients previously treated with ceftolozane/tazobactam (73%; 8/11). Isolates with reduced cefiderocol susceptibility harboured mutations in ampC, tonB-dependent receptors, the response regulator pirR and ftsI. Cefiderocol demonstrates excellent in vitro activity against P. aeruginosa isolates exposed to other novel β-lactam agents; however, some exceptions were identified. Cross-resistance between cefiderocol and ceftolozane/tazobactam was evident, but not with ceftazidime/avibactam or imipenem/relebactam. Reduced cefiderocol susceptibility was mediated by mutations in ampC and tonB-dependent receptors.

Evaluation of Metallo-β-Lactamase Susceptibility Testing in a Physiologic Medium.

ABSTRACTResearch in identifying alternative growth media that better mimic host conditions is gaining ground. Relative to nutrient-rich Mueller-Hinton broth (MHB), data on the influence of physiologic or host-mimicking media on metallo-β-lactamase (MBL) resistance are lacking. The objective was to evaluate meropenem susceptibility against clinical and engineered MBL-harboring Enterobacterales strains in a physiologic medium (urine). Antimicrobial susceptibility testing (AST) by broth microdilution was conducted with a wild-type Klebsiella pneumoniae strain and two engineered isogenic variants harboring K. pneumoniae carbapenemase 2 (KPC-2) or New Delhi MBL 1 (NDM-1), as well as two clinical K. pneumoniae isolates (harboring NDM-1 and VIM-1). MICs were determined in conventional cation-adjusted MHB (caMHB) and sterile-filtered urine samples (18 patients). All KPC- and MBL-harboring isolates were meropenem resistant (MICs of ≥16 mg/liter) in caMHB. AST of the KPC isolate in urine resulted in 50% (9/18 urine samples) essential agreement (i.e., within ±1 dilution, relative to the caMHB MIC), highlighting challenges with the use of urine as a medium capable of supporting AST. In the 9 AST-viable urine samples, meropenem MICs were 2- to 9-fold lower than that in caMHB (MIC of 32 mg/liter) among MBL-harboring isolates. Zinc concentrations determined by inductively coupled plasma mass spectrometry averaged 1.25 mg/liter and ranged from 0.12 to 1.14 mg/liter in caMHB and 18 urine samples, respectively. The full extent of MBL-mediated resistance among K. pneumoniae isolates appears to be attenuated in urine. Factors influencing free bioactive zinc levels warrant further investigation.IMPORTANCE Studies assessing antibiotic susceptibility profiles in nonconventional media are lacking. MBL-mediated resistance has come under scrutiny due to the dependence on extracellular zinc concentrations, which makes the choice of testing medium influential for β-lactam MICs. This study explores human urine as a physiologically relevant matrix with which susceptibility profiles of MBL-harboring isolates can be assessed, relative to conventional broth.

KPC-2-Producing Carbapenem-Resistant Klebsiella pneumoniae of the Uncommon ST29 Type Carrying OXA-926, a Novel Narrow-Spectrum OXA β-Lactamase.

We isolated and characterized a carbapenem-resistant Klebsiella pneumoniae (CRKP) clinical strain from blood carrying a novel blaOXA gene, blaOXA–926, and belonging to ST29, an uncommon CRKP type. The strain, 130002, was genome sequenced using both short- and long-read sequencing and has a 94.9-kb self-transmissible IncFII plasmid carrying blaKPC–2. K. pneumoniae genomes of the ST29 complex (ST29 and its single-allele variants) were retrieved and were subjected to single nucleotide polymorphism-based phylogenomic analysis. A total of 157 genomes of the ST29 complex were identified. This complex is commonly associated with extended-spectrum β-lactamase-encoding genes, in particular, blaCTX–M–15 but rarely has carbapenemase genes. The novel plasmid-encoded β-lactamase-encoding gene blaOXA–926 was identified on a 117.8-kb IncFIA-IncFII plasmid, which was transferrable in the presence of the blaKPC–2-carrying plasmid. blaOXA–926 was cloned and MICs of β-lactams in the transformants were determined using microdilution. OXA-926 has a narrow spectrum conferring reduced susceptibility only to piperacillin, piperacillin-tazobactam, and cephalothin. Avibactam cannot fully inhibit OXA-926. blaOXA–926 and its variants have been seen in Klebsiella strains in Asia and Brazil. OXA-926 is the closest in sequence identity (89.9%) to a chromosome-encoding OXA-type enzyme of Variovorax guangxiensis. In conclusion, OXA-926 is novel plasmid-borne narrow-spectrum β-lactamase that cannot be fully inhibited by avibactam. It is likely that blaOXA–926 originates from a species closely related to V. guangxiensis and was introduced into Klebsiella > 10 years ago.

CON: Testing for ESBL production is unnecessary for ceftriaxone-resistant Enterobacterales.

Phenotypic testing for Enterobacterales that harbour ESBLs is not additive to accurate in vitro β-lactam MICs for clinical decision-making. ESBL testing is an outdated practice established in an era of higher cephalosporin breakpoints to prevent resistant Enterobacterales carrying Ambler class A β-lactamases with affinity for later-generation β-lactams from being reported as susceptible to later-generation cephalosporins, leading to clinical failures. ESBL testing is problematic because of inaccuracies when multiple classes of β-lactamases are produced by the same organism, thus limiting the testing application to specific species and resistance types. Clinical laboratories should instead focus finite resources on accurate susceptibility testing using contemporary interpretative criteria to help guide therapeutic decisions. With continued emergence of antimicrobial resistance and in the setting of accurate susceptibility testing and current breakpoints the use of ESBL phenotypic testing is not helpful in clinical decision-making.

Optimizing pharmacokinetics/pharmacodynamics of β-lactam/β-lactamase inhibitor combinations against high inocula of ESBL-producing bacteria

Reduced in vitro β-lactam activity against a dense bacterial population is well recognized. It is commonly attributed to the presence of β-lactamase(s) and it is unknown whether the inoculum effect could be diminished by a β-lactamase inhibitor. We evaluated different β-lactam/β-lactamase inhibitor combinations in suppressing a high inoculum of ESBL-producing bacteria. Three clinical isolates expressing representative ESBLs (CTX-M-15 and SHV-12) were examined. The impact of escalating β-lactamase inhibitor (tazobactam or avibactam) concentrations on β-lactam (piperacillin or ceftazidime) MIC reduction was characterized by an inhibitory sigmoid Emax model. The effect of various dosing regimens of β-lactam/β-lactamase inhibitor combinations was predicted using %T>MICi and selected exposures were experimentally validated in a hollow-fibre infection model over 120 h. The threshold exposure to suppress bacterial regrowth was identified using recursive partitioning. A concentration-dependent reduction in β-lactam MIC was observed (r2 ≥0.93). Regrowth could be suppressed in all six experiments using %T>MICi ≥73.6%, but only one out of six experiments below the threshold (P = 0.015). The exposures to suppress regrowth might be attained using the clinical dose of avibactam, but a much higher dose than the standard dose would be needed for tazobactam. A dense population of ESBL-producing bacteria could be suppressed by an optimized dosing regimen of selected β-lactam/β-lactamase inhibitor combinations. The reversibility of enzyme inhibition could play an important role in diminishing the inoculum effect. In vivo investigations to validate these findings are warranted.

A Novel KPC Variant KPC-55 in Klebsiella pneumoniae ST307 of Reinforced Meropenem-Hydrolyzing Activity.

A novel Klebsiella pneumoniae carbapenemase (KPC) variant, KPC-55, produced by a K. pneumoniae ST307 strain was characterized. K. pneumoniae strain BS407 was recovered from an active surveillance rectal swab of a patient newly admitted to a general hospital in Busan, South Korea. Carbapenemase production was confirmed by the modified Hodge test, and the MICs of β-lactams were determined by the broth microdilution method. The whole genome was sequenced. Cloning and expression of the blaKPC–55 gene in Escherichia coli and MIC determination were performed. The enzyme KPC-55 was used for kinetic assays against β-lactams and compared with the KPC-2 enzyme. The new allele of the blaKPC gene had a T794A alteration compared to the blaKPC–2 gene, resulting in the amino acid substitution Y264N in the middle of the β9-sheet. Compared to the KPC-2-producing strain, the KPC-55-producing strain exhibited a lower level of resistance to most β-lactam drugs tested, however, the KPC-55 enzyme catalyzed aztreonam and meropenem at an increased efficiency compared to the catalytic activity of KPC-2. KPC subtypes could have varied phenotypes due to alterations in amino acid sequences, and such an unexpected resistance phenotype emphasizes the importance of detailed characterizations for the carbapenemase-producing Enterobacterales.

Dalbavancin exposure in vitro selects for dalbavancin-non-susceptible and vancomycin-intermediate strains of methicillin-resistant Staphylococcus aureus

ObjectivesDalbavancin is a lipoglycopeptide active against methicillin-resistant Staphylococcus aureus (MRSA). Its long half-life (8.5–16 days) allows for once-weekly or single-dose treatments but could prolong the mutant selection window, promoting resistance and cross-resistance to related antimicrobials such as vancomycin. The objective of this study was to evaluate the capacity of post-distributional pharmacokinetic exposures of dalbavancin to select for resistance and cross-resistance in MRSA. MethodsWe simulated average, post-distributional exposures of single-dose (1500 mg) dalbavancin (fCmax 9.9 μg/mL, β-elimination t1/2 204 h) in an in vitro pharmacokinetic/pharmacodynamic (PK/PD) model for 28 days (672 h) against five MRSA strains and one methicillin-susceptible strain (MSSA). Samples were collected at least daily, and surviving colonies were enumerated and screened for resistance on drug-free and dalbavancin-supplemented medium respectively. Isolates from resistance screening plates were subjected to whole-genome sequencing (WGS) and susceptibly testing against dalbavancin, vancomycin, daptomycin, and six β-lactams with varying penicillin-binding protein (PBP) affinities. ResultsDalbavancin was bactericidal against most strains for days 1–4 before regrowth of less susceptible subpopulations occurred. Isolates with eight-fold increases in dalbavancin MIC were detected as early as day 4 but increased 64–128-fold in all models by day 28. Vancomycin and daptomycin MICs increased 4–16-fold, exceeding the susceptibly breakpoints for both antibiotics; β-lactam MICs generally decreased by two-to eight-fold, suggesting a dalbavancin–β-lactam seesaw effect, but increased by eight-fold or more in certain isolates. Resistant isolates carried mutations in a variety of genes, most commonly walKR, apt, stp1, and atl. ConclusionsIn our in vitro system, post-distributional dalbavancin exposures selected for stable mutants with reduced susceptibility to dalbavancin, vancomycin, and daptomycin, and generally increased susceptibility to β-lactams in all strains of MRSA tested. The clinical significance of these findings remains unclear, but created an opportunity to genotype a unique collection of dalbavancin-resistant strains for the first time. Mutations involved genes previously associated with vancomycin intermediate susceptibility and daptomycin non-susceptibility, most commonly walKR-associated genes.

Scope and Predictive Genetic/Phenotypic Signatures of Bicarbonate (NaHCO3) Responsiveness and β-Lactam Sensitization in Methicillin-Resistant Staphylococcus aureus.

Addition of sodium bicarbonate (NaHCO3) to standard antimicrobial susceptibility testing medium reveals certain methicillin-resistant Staphylococcus aureus (MRSA) strains to be highly susceptible to β-lactams. We investigated the prevalence of this phenotype (NaHCO3 responsiveness) to two β-lactams among 58 clinical MRSA bloodstream isolates. Of note, ∼75% and ∼36% of isolates displayed the NaHCO3 responsiveness phenotype to cefazolin (CFZ) and oxacillin (OXA), respectively. Neither intrinsic β-lactam MICs in standard Mueller-Hinton broth (MHB) nor population analysis profiles were predictive of this phenotype. Several genotypic markers (clonal complex 8 [CC8]; agr I and spa t008) were associated with NaHCO3 responsiveness for OXA.

Antibiotic Susceptibility of Haemophilus influenzae in Sfax: Two Years after the Introduction of the Hib Vaccination in Tunisia

A retrospective study was conducted at the Sfax hospitals between 2013 and 2014 to study the susceptibility to antibiotics of Haemophilus influenzae after the introduction of Hib vaccination in Tunisia. Capsular typing was done by PCR. MICs for β-lactams were determined by E-test®. The blaTEM, blaROB and ftsI genes were searched using PCR. Among the 259 strains of H. influenzae isolated, 248 (95.7%) were non-invasive. Five strains were encapsulated (3 type b and 2 type c). Resistance rates were 33.4% for ampicillin, 10.4% for amoxicillin-clavulanic acid, 2.3% for cefixime, 1.1% for cefotaxime and 1.9% for fluoroquinolones. Among the strains resistant to β-lactams, 67 produced β-lactamase type TEM and 40 had a modification of PLP3. This study shows the change in the epidemiology of H. influenzae induced by vaccination with a dramatic decrease of invasive infections. Replacement with other capsular types or with non-typable strains that may be resistant to antibiotics requires continuous surveillance for H. influenzae infections.

Occurrence of cross-resistance and β-lactam seesaw effect in glycopeptide-, lipopeptide- and lipoglycopeptide-resistant MRSA correlates with membrane phosphatidylglycerol levels.

Glycopeptides (GPs), lipopeptides (LPs) and lipoglycopeptides (LGPs) are related antimicrobials important for the management of invasive MRSA infections. Cross-resistance among these antibiotics in MRSA is well documented, as is the observation that susceptibility of MRSA to β-lactams increases as susceptibility to GPs and LPs decreases (i.e. the seesaw effect). Efforts to understand the relationship between GP/LP/LGP cross-resistance and the seesaw effect have focused on the PBPs, but the role of lipid metabolism has not been investigated. Since the cell membrane is structurally and metabolically integrated with the cell wall and anchors associated proteins, including PBPs, we examined the relationship between membrane lipid composition and the phenomena of cross-resistance among GPs/LPs/LGPs and the β-lactam seesaw effect. We selected for daptomycin, vancomycin and dalbavancin resistance using the USA300 strain JE2 and evaluated the resulting mutants by WGS, MS-based lipidomics and antimicrobial susceptibility testing to assess the relationship between membrane composition, cross-resistance, and the seesaw effect. We observed cross-resistance to GPs/LPs/LGPs among the selected strains and the seesaw effect against various β-lactams, depending on the PBP targets of the particular β-lactam. We found that modification of membrane composition occurs not only in daptomycin-selected strains, but also vancomycin- and dalbavancin-selected strains. Significantly, we observed that the abundance of most phosphatidylglycerols positively correlates with MICs of GPs/LPs/LGPs and negatively correlates with the MICs of β-lactams. These studies demonstrate a major association between membrane remodelling, cross-resistance and the seesaw effect.

Mechanisms of β-lactam resistance of Streptococcus uberis isolated from bovine mastitis cases

A number of veterinary clinical pathology laboratories in New Zealand have been reporting emergence of increased minimum in inhibitory concentrations for β-lactams in the common clinical bovine mastitis pathogen Streptococcus uberis. The objective of this study was to determine the genetic basis of this increase in MIC for β-lactams amongst S. uberis. Illumina sequencing and determination of oxacillin MIC was performed on 265 clinical isolates. Published sequences of the five penicillin binding proteins pbp1a, pbp1b, pbp2a, pbp2b, and pbp2x were used to identify, extract and align these sequences from the study isolates. Amino acid substitutions resulting from single nucleotide polymorphisms (SNP) within these genes were analysed for associations with elevated (≥ 0.5 mg/L) oxacillin MIC together with a genome wide association study. The population structure of the study isolates was approximated using a phylogenetic tree generated from an alignment of the core genome. A total of 53 % of isolates had MIC ≥ 0.5 mg/L for oxacillin. A total of 101 substitutions within the five pbp were identified, of which 11 were statistically associated with an MIC ≥ 0.5 mg/L. All 140 isolates which exhibited an increased β-lactam MIC had SNPs leading to pbp2x E381K and Q554E substitutions. The phylogenetic tree indicated that the genotype and phenotype associated with the increased MIC for oxacillin were present in several different lineages suggesting that acquisition of this increased β-lactam MIC had occurred in multiple geographically distinct regions. Reanalysis of the data from the intervention studies from which the isolates were originally drawn found a tendency for the pbp2x E381K substitution to be associated with lower cure rates. It is concluded that there is geographically and genetically widespread presence of pbp substitutions associated with reduced susceptibility to β-lactam antimicrobials. Additionally, presence of pbp substitutions tended to be associated with poorer cure rate outcomes following antimicrobial therapy for clinical mastitis.

Improved β-Lactam Susceptibility Against ica-Dependent Biofilm-Embedded Staphylococcus aureus by 2-Aminothiazole.

Due to the emergence of methicillin-resistant Staphylococcus aureus (MRSA) producing biofilm, causing recurrent infections worldwide, new therapeutic combinations need to be discovered to prevent resistance and to make treatment available. It was aimed to improve β-lactam susceptibility against ica-dependent biofilm-embedded Staphylococcus aureus (S. aureus), even in blaZ and mecA carriers, by 2-aminothiazole. Virulence genes in isolates were detected by qRT-PCR. MICs, MBCs of 2-aminothiazole and β-lactams against planktonic and sessile ica-dependent biofilm-producer isolates were investigated. Activities of 2-aminothiazole combined β-lactams against sessile one MRSA ATCC 43300, one MRSA, and two MSSA were investigated by checkerboard-assay. Activities of 2-aminothiazole combined β-lactams were found synergistic and partially-synergistic against both biofilm-embedded MRSA and MSSA-isolates with FIC-indexes ranging between 0.193 - 0.387 and 0.535 - 0.745 and between 0.358 - 0.415 and 0.707 - 1.0, respectively. MICs of β-lactams against MSSA and MRSA were decreased 2- to 8-fold and 0- to 8-fold by sub-MICs of 2-aminothiazole, respectively. Sub-MIC 2-aminothiazole combined Sub-MIC β-lactams can be a choice in varying applications to treat biofilm-associated staphylococcal infections.

Lower respiratory tract isolates of non-typeable Haemophilus influenzae in Western Sichuan, China: Antimicrobial susceptibility, mechanisms of β-lactam resistance and decade changes

ObjectivesThe aims of this study were to analyse the serotypes of epidemic Haemophilus influenzae and changes in mechanisms of β-lactam resistance over the past decade. ResultsHaemophilus influenzae isolates in Western Sichuan from 2013–2014 were non-typeable H. influenzae (NTHi). β-Lactam MICs for NTHi isolated during 2013–2014 were significantly higher than those from 2003–2004 (P < 0.05). Of 274 NTHi, 141 (51.5%) were β-lactamase-positive (TEM-1 type). There were 35 amino acid (AA) substitutions in ftsI among NTHi isolated from 2013–2014. However, NTHi isolates from 2003–2004 had only nine AA substitutions. Ordered multiple classification logistic regression analysis showed that different AA substitution patterns in ftsI had different effects on β-lactam MICs. The main factors affecting the ampicillin MIC were the mutations R517H (OR = 6.999), L389F (OR = 7.128), N526K (OR = 4.660) and D350N (OR = 0.450). The main factor influencing the amoxicillin/clavulanic acid MIC was an N526K mutation (OR = 9.349). The main factors affecting the cefuroxime MIC were the mutations S357N (OR = 37.453) and N526K (OR = 14.816). Compared with 2003–2004, gBLNAR and gBLPAR isolated from 2013–2014 increased significantly from 13.0% (7/54) and 9.3% (5/54) to 38.2% (84/220) and 45.5% (100/220), respectively (P < 0.001). In the ‘others’ group of ftsI gene mutations, 13 NTHi had the same ftsI gene mutation pattern and 24 AA substitutions. ConclusionThese results confirm that β-lactam-resistant NTHi isolates increased rapidly. AA substitutions in ftsI were more complex and diversified in 2013–2014.

1600. Susceptibility of β-Lactam-Resistant Pseudomonas aeruginosa to Other β-Lactams: Is There Truly a Lack of Cross-Resistance?

BackgroundResistance to β-lactams in P. aeruginosa is complex with multiple mechanisms contributing. Since different mechanisms impact different β-lactams to differing degrees, a common dogma is that resistance to one β-lactam does not lead to resistance to others. The purpose of this analysis was to assess the frequency of β-lactam cross-resistance in P. aeruginosa.MethodsUnique P. aeruginosa isolated in 2017 at Michigan Medicine were included. Overall, susceptibility (using CLSI breakpoints) and MIC distributions of β-lactams were assessed in all isolates and those with β-lactam resistance.Results3,836 unique P. aeruginosa isolates were included. Resistance to traditional anti-pseudomonal β-lactams ranged from 15–23%, whereas ceftolozane/tazobactam resistance was 6%. Overall, cross-resistance between β-lactams was common. The table displays select β-lactam MIC distributions for all isolates and in those resistant to ≥1 β-lactam. When resistance of one agent was present susceptibility to other β-lactams was generally <40% with the majority of susceptible isolates having MICs at or near the breakpoint. Ceftolozane/tazobactam provided the best activity in this setting with 65–77% susceptibility.ConclusionCross-resistance between β-lactams in P. aeruginosa is common. In patients at risk for resistant P. aeruginosa, ceftolozane/tazobactam should be considered for empiric coverage. Disclosures All authors: No reported disclosures.

Characterization of mutations in Escherichia coli PBP2 leading to increased carbapenem MICs.

To examine the impact on carbapenem resistance of mutations in Escherichia coli PBP2 detected in clinical isolates showing increased MICs of imipenem, but not of meropenem. The mutations in the PBP2-encoding gene mrdA were introduced into E. coli DH5α using the helper plasmid pTKRED. β-Lactam MICs were determined by broth microdilution and interpreted according to EUCAST. Mutants were screened for secondary mutations by WGS. To detect a possible fitness cost related to these mutations, the generation times of the mutants and E. coli DH5α were measured and their cell morphology was examined. The 10 mutation patterns introduced into mrdA increased the MICs of imipenem and doripenem in all cases and of meropenem and mecillinam in some cases, but had no effect on ertapenem resistance. While no significant alteration of the generation time of the mutants could be detected, several mutants showed increased transverse diameters and thus altered cell morphology. Here we describe mutation patterns in PBP2 that contribute to increased MICs of carbapenems for clinical isolates of E. coli. We show that different mutations affect carbapenem MICs differently and that the mutations do not impact generation time, although some mutations affect cell morphology.

Evolution of the β-lactam-resistant Streptococcus pneumoniae PMEN3 clone over a 30 year period in Barcelona, Spain.

To analyse the epidemiology and genetic evolution of PMEN3 (Spain9V-156), a penicillin-non-susceptible clone of Streptococcus pneumoniae, causing invasive pneumococcal disease (IPD) in Barcelona during 1987-2016. WGS was performed on 46 representative isolates and the data were used to design additional molecular typing methods including partial MLST, PCR-RFLP and detection of surface-exposed proteins and prophages, to assign the remaining isolates to lineages. The isolates were also subjected to antimicrobial susceptibility testing. Two hundred and twenty-seven adult cases of IPD caused by PMEN3 were identified. PMEN3 caused mainly pneumonia (84%) and the 30 day mortality rate was 23.1%. Evidence of recombination events was found, mostly in three regions, namely the capsular operon (associated with capsular switching) and adjacent regions containing pbp2x and pbp1a, the murM gene and the pbp2b-ddl region. Some of these genetic changes generated successful new variant serotype lineages, including one of serotype 11A that is not included in the current PCV13 vaccine. Other genetic changes led to increased MICs of β-lactams. Notably, most isolates also harboured prophages coding for PblB-like proteins. Despite these adaptations, the ability of this clone to cause IPD remained unchanged over time, highlighting the importance of its core genetic background. Our study demonstrated successful adaptation of PMEN3 to persist over time despite the introduction of broader antibiotics and conjugate vaccines. In addition to enhancing understanding of the molecular evolution of PMEN3, these findings highlight the need for the development of non-serotype-based vaccines to fight pneumococcal infection.

Carbapenem-Nonsusceptible Haemophilus influenzae with Penicillin-Binding Protein 3 Containing an Amino Acid Insertion.

The prevalence of β-lactamase-negative ampicillin-resistant (BLNAR) Haemophilus influenzae has become a clinical concern. In BLNAR isolates, amino acid substitutions in penicillin-binding protein 3 (PBP3) are relevant to the β-lactam resistance. Carbapenem-nonsusceptible H. influenzae isolates have been rarely reported. Through antimicrobial susceptibility testing, nucleotide sequence analysis of ftsI, encoding PBP3, and the utilization of a collection of H. influenzae clinical isolates in our laboratory, we obtained a carbapenem-nonsusceptible clinical isolate (NUBL1772) that possesses an altered PBP3 containing V525_N526insM. The aim of this study was to reveal the effect of altered PBP3 containing V525_N526insM on reduced carbapenem susceptibility. After generating recombinant strains with altered ftsI, we performed antimicrobial susceptibility testing and competitive binding assays with fluorescent penicillin (Bocillin FL) and carbapenems. Elevated carbapenem MICs were found for the recombinant strain harboring the entire ftsI gene of NUBL1772. The recombinant PBP3 of NUBL1772 also exhibited reduced binding to carbapenems. These results demonstrate that altered PBP3 containing V525_N526insM influences the reduced carbapenem susceptibility. The revertant mutant lacking the V525_N526insM exhibited lower MICs for carbapenems than NUBL1772, suggesting that this insertion affects reduced carbapenem susceptibility. The MICs of β-lactams for NUBL1772 were higher than those for the recombinant possessing ftsI of NUBL1772. NUBL1772 harbored AcrR with early termination, resulting in low-level transcription of acrB and high efflux pump activity. These findings suggest that the disruption of AcrR also contributes to the reduced carbapenem susceptibility found in NUBL1772. Our results provide the first evidence that the altered PBP3 containing V525_N526insM is responsible for the reduced susceptibility to carbapenems in H. influenzae.

Unusual Escherichia coli PBP 3 Insertion Sequence Identified from a Collection of Carbapenem-Resistant Enterobacteriaceae Tested In Vitro with a Combination of Ceftazidime-, Ceftaroline-, or Aztreonam-Avibactam.

Carbapenemase-producing Enterobacteriaceae isolates (n = 110) from health care centers in central Indiana (from 2010 to 2013) were tested for susceptibility to combinations of avibactam (4 μg/ml) with ceftazidime, ceftaroline, or aztreonam. MIC50/MIC90 values were 1/2 μg/ml (ceftazidime-avibactam), 0.5/2 μg/ml (ceftaroline-avibactam), and 0.25/0.5 μg/ml (aztreonam-avibactam.) A β-lactam MIC of 8 μg/ml was reported for the three combinations against one Escherichia coli isolate with an unusual TIPY insertion following Tyr344 in penicillin-binding protein 3 (PBP 3) as the result of gene duplication.

Penicillin-Binding Protein Transpeptidase Signatures for Tracking and Predicting β-Lactam Resistance Levels in Streptococcus pneumoniae.

ABSTRACTβ-Lactam antibiotics are the drugs of choice to treat pneumococcal infections. The spread of β-lactam-resistant pneumococci is a major concern in choosing an effective therapy for patients. Systematically tracking β-lactam resistance could benefit disease surveillance. Here we developed a classification system in which a pneumococcal isolate is assigned to a “PBP type” based on sequence signatures in the transpeptidase domains (TPDs) of the three critical penicillin-binding proteins (PBPs), PBP1a, PBP2b, and PBP2x. We identified 307 unique PBP types from 2,528 invasive pneumococcal isolates, which had known MICs to six β-lactams based on broth microdilution. We found that increased β-lactam MICs strongly correlated with PBP types containing divergent TPD sequences. The PBP type explained 94 to 99% of variation in MICs both before and after accounting for genomic backgrounds defined by multilocus sequence typing, indicating that genomic backgrounds made little independent contribution to β-lactam MICs at the population level. We further developed and evaluated predictive models of MICs based on PBP type. Compared to microdilution MICs, MICs predicted by PBP type showed essential agreement (MICs agree within 1 dilution) of >98%, category agreement (interpretive results agree) of >94%, a major discrepancy (sensitive isolate predicted as resistant) rate of <3%, and a very major discrepancy (resistant isolate predicted as sensitive) rate of <2% for all six β-lactams. Thus, the PBP transpeptidase signatures are robust indicators of MICs to different β-lactam antibiotics in clinical pneumococcal isolates and serve as an accurate alternative to phenotypic susceptibility testing.