Beta-lactamase dependent and independent evolutionary paths to high-level ampicillin resistance.

The incidence of beta-lactam resistance among clinical isolates is a major health concern. A key method to study the emergence of antibiotic resistance is adaptive laboratory evolution. However, in the case of the beta-lactam ampicillin, bacteria evolved in laboratory settings do not recapitulate clinical-like resistance levels, hindering efforts to identify major evolutionary paths and their dependency on genetic background. Here, we used the Microbial Evolution and Growth Arena (MEGA) plate to select ampicillin-resistant Escherichia coli mutants with varying degrees of resistance. Whole-genome sequencing of resistant isolates revealed that ampicillin resistance was acquired via a combination of single-point mutations and amplification of the gene encoding beta-lactamase AmpC. However, blocking AmpC-mediated resistance revealed latent adaptive pathways: strains deleted for ampC were able to adapt through combinations of changes in genes involved in multidrug resistance encoding efflux pumps, transcriptional regulators, and porins. Our results reveal that combinations of distinct genetic mutations, accessible at large population sizes, can drive high-level resistance to ampicillin even independently of beta-lactamases.

Ampicillin susceptibility testing of Haemophilus influenzae in the routine clinical laboratory by the EUCAST methodology compared to broth microdilution and the presence of ftsI gene mutations.

OBJECTIVES: To investigate the prevalence of ampicillin resistance in Haemophilus influenzae and the diagnostic accuracy of the EUCAST recommended disc diffusion method to detect the increasingly prevalent ampicillin resistance due to the presence of PBP3 alterations based on mutations in the ftsI gene. METHODS: During a 6-month period all consecutive non-duplicate H. influenzae isolates were prospectively collected and stored. MICs of ampicillin were determined by broth microdilution (BMD). PCR was performed to detect mutations in the ftsI gene. Results of routine disc diffusion susceptibility testing, including the penicillin screening test in accordance with the current EUCAST methodology, as well as additional Etest results, were compared to the BMD as the reference method. RESULTS: In 102 isolates, the prevalence of ampicillin resistance was 28% (29/102) by BMD. There was a good correlation between MICs of ampicillin and the presence of a ß-lactamase and/or an ftsI gene mutation. The prevalence of ampicillin resistance was overestimated using the EUCAST method (33% (34/102)) and underestimated when an additional Etest was used (24% (24/102)) (not significant). The sensitivity and specificity of the EUCAST methodology for the detection of ampicillin resistance were 97% ((28/29); 95% CI, 82-100%) and 92% ((67/73); 95% CI, 83-97%), respectively. CONCLUSIONS: The prevalence of ampicillin resistance was 28%, as determined by BMD. Although the overall diagnostic accuracy of the EUCAST ampicillin disc diffusion was high, misclassification of ampicillin susceptibility may still occur.

Resistance mechanism of Escherichia coli strains with different ampicillin resistance levels.

Antibiotic resistance is an important problem that threatens medical treatment. Differences in the resistance levels of microorganisms cause great difficulties in understanding the mechanisms of antibiotic resistance. Therefore, the molecular reasons underlying the differences in the level of antibiotic resistance need to be clarified. For this purpose, genomic and transcriptomic analyses were performed on three Escherichia coli strains with varying degrees of adaptive resistance to ampicillin. Whole-genome sequencing of strains with different levels of resistance detected five mutations in strains with 10-fold resistance and two additional mutations in strains with 95-fold resistance. Overall, three of the seven mutations occurred as a single base change, while the other four occurred as insertions or deletions. While it was thought that 10-fold resistance was achieved by the effect of mutations in the ftsI, marAR, and rpoC genes, it was found that 95-fold resistance was achieved by the synergistic effect of five mutations and the ampC mutation. In addition, when the general transcriptomic profiles were examined, it was found that similar transcriptomic responses were elicited in strains with different levels of resistance. This study will improve our view of resistance mechanisms in bacteria with different levels of resistance and provide the basis for our understanding of the molecular mechanism of antibiotic resistance in ampicillin-resistant E. coli strains. KEY POINTS: •The mutation of the ampC promoter may act synergistically with other mutations and lead to higher resistance. •Similar transcriptomic responses to ampicillin are induced in strains with different levels of resistance. •Low antibiotic concentrations are the steps that allow rapid achievement of high antibiotic resistance.

Use of trans-complementation method to determine the effects of various ftsI mutations on ß-lactamase-negative ampicillin-resistant (BLNAR) Haemophilus influenzae strains.

Haemophilus influenzae is a causative agent of serious infections, especially among children. ß-lactam antibiotics are commonly used for the treatment of these infections. Among H. influenzae isolates, ß-lactam resistance is due to the presence of ß-lactamase, or to mutations in the ftsI gene that generate altered PBP3 (penicillin-binding protein 3) with reduced affinity for ß-lactams (BLNAR-ß-lactamase-negative, ampicillin-resistant). Wild-type ftsI gene encoding for PBP3 was amplified in whole from ß-lactam susceptible H. influenzae Rd and cloned in pLS88 plasmid to obtain pADUTAS17, which was then used to transform known BLNAR strains, susceptible strains, and a strain (CF55) with wild-type ftsI but unexplained reduced ß-lactam susceptibility. Ampicillin and cefotaxime MICs (minimum inhibitory concentration) were determined after transformation with pLS88 and pADUTAS17 plasmids. The results showed that antibiotic susceptibilities were not affected by trans-complementation for isolates carrying wild-type ftsI gene. However, trans-complementation for all BLNAR strains showed decreases between - 0.957 and 0.5-fold for ampicillin and cefotaxime, confirming the role of the PBP3 substitutions in the BLNAR phenotype of these isolates. The first article showed that trans-complementation might be a useful tool in the investigation of decreased ß-lactam susceptibility in H. influenzae.

Metabolic phenotyping of acquired ampicillin resistance using microbial volatiles from Escherichia coli cultures.

AIMS: This study sought to assess the volatile organic compound (VOC) profiles of ampicillin-resistant and -susceptible Escherichia coli to evaluate whether VOC analysis may be utilized to identify resistant phenotypes. METHODS AND RESULTS: An E. coli BL21 (DE3) strain and its pET16b plasmid transformed ampicillin-resistant counterpart were cultured for 6 h in drug-free, low- and high-concentrations of ampicillin. Headspace analysis was undertaken using thermal desorption-gas chromatography-mass spectrometry. Results revealed distinct VOC profiles with ampicillin-resistant bacteria distinguishable from their susceptible counterparts using as few as six compounds. A minimum of 30 compounds (fold change >2, p ≤ 0.05) were differentially expressed between the strains across all set-ups. Furthermore, three compounds (indole, acetoin and 3-methyl-1-butanol) were observed to be significantly more abundant (fold change >2, p ≤ 0.05) in the resistant strain compared to the susceptible strain both in the presence and in the absence of drug stress. CONCLUSIONS: Results indicate that E. coli with acquired ampicillin resistance exhibit an altered VOC profile compared to their susceptible counterpart both in the presence and in the absence of antibiotic stress. This suggests that there are fundamental differences between the metabolisms of ampicillin-resistant and -susceptible E. coli which may be detected by means of VOC analysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings suggest that VOC profiles may be utilized to differentiate between resistant and susceptible bacteria using just six compounds. Consequently, the development of machine-learning models using VOC signatures shows considerable diagnostic applicability for the rapid and accurate detection of antimicrobial resistance.

Dysregulation of Streptococcus pneumoniae zinc homeostasis breaks ampicillin resistance in a pneumonia infection model.

Streptococcus pneumoniae is the primary cause of community-acquired bacterial pneumonia with rates of penicillin and multidrug-resistance exceeding 80% and 40%, respectively. The innate immune response generates a variety of antimicrobial agents to control infection, including zinc stress. Here, we characterize the impact of zinc intoxication on S. pneumoniae, observing disruptions in central carbon metabolism, lipid biogenesis, and peptidoglycan biosynthesis. Characterization of the pivotal peptidoglycan biosynthetic enzyme GlmU indicates a sensitivity to zinc inhibition. Disruption of the sole zinc efflux pathway, czcD, renders S. pneumoniae highly susceptible to ß-lactam antibiotics. To dysregulate zinc homeostasis in the wild-type strain, we investigated the safe-for-human-use ionophore 5,7-dichloro-2-[(dimethylamino)methyl]quinolin-8-ol (PBT2). PBT2 rendered wild-type S. pneumoniae strains sensitive to a range of antibiotics. Using an invasive ampicillin-resistant strain, we demonstrate in a murine pneumonia infection model the efficacy of PBT2 + ampicillin treatment. These findings present a therapeutic modality to break antibiotic resistance in multidrug-resistant S. pneumoniae.

An update on ampicillin resistance and ß-lactamase genes of Bacteroides spp.

Introduction. There are several ß-lactamase genes described for Bacteroides strains, of which cepA and cfiA are specific for Bacteroides fragilis and define two genetic divisions. The expression and phenotypic effects of these genes are usually regulated by insertional activation.Hypotheses/Gap Statement. Information is lacking about how cepA is regulated for most of the B. fragilis strains and whether there could be a genetic element for it.Aim. We aimed to investigate the molecular background of ampicillin (and other ß-lactam) resistance among Bacteroides strains as mediated mainly by cepA and also to find a genetic element for it as known for cfiA.Methodology. Various PCR methods were used for ß-lactamase-resistance gene and insertion sequence (IS) element detection in 42 Bacteroides strains. ß-Lactamase activity measurements and antimicrobial-susceptibility testing using agar dilution were also applied. Further molecular experiments involved sequencing, gene targeting, Southern blotting and bioinformatic analyses.Results. We found that high antibiotic resistance and ß-lactamase levels are brought about by insertional activation of the cepA gene or by similar or dissimilar activation of cfxA or cfiA, or by the newly described pbbA genes. Non-activated cepA genes produced low levels of specific ß-lactamase activities that did not correlate with ampicillin resistance. We found a genetic element for cepA and another region close to it that are characteristic for division I B. fragilis strains, which are replaced by other sequences in division II B. fragilis strains.Conclusion. cepA usually is not activated by IS elements and usually produces low ß-lactamase activities that do not correlate with the ampicillin MICs; therefore, it probably involves some non-ß-lactamase-mediated resistance mechanism(s). pbpA is a newly described, effective ß-lactamase gene that is located on a plasmid, and cepA resides on a well-defined chromosomal segment that is mutually replaced in division II B. fragilis strains. This latter finding demonstrates the genetic dichotomy of cepA-cfiA in B. fragilis and requires further investigation.

Efficient association mapping from k-mers-An application in finding sex-specific sequences.

Genome wide association studies (GWAS) attempt to map genotypes to phenotypes in organisms. This is typically performed by genotyping individuals using microarray or by aligning whole genome sequencing reads to a reference genome. Both approaches require knowledge of a reference genome which hinders their application to organisms with no or incomplete reference genomes. This caveat can be removed by using alignment-free association mapping methods based on k-mers from sequencing reads. Here we present an improved implementation of an alignment free association mapping method. The new implementation is faster and includes additional features to make it more flexible than the original implementation. We have tested our implementation on an E. Coli ampicillin resistance dataset and observe improvement in execution time over the original implementation while maintaining accuracy in results. We also demonstrate that the method can be applied to find sex specific sequences.

A Cas-embedding strategy for minimizing off-target effects of DNA base editors.

DNA base editors, typically comprising editing enzymes fused to the N-terminus of nCas9, display off-target effects on DNA and/or RNA, which have remained an obstacle to their clinical applications. Off-target edits are typically countered via rationally designed point mutations, but the approach is tedious and not always effective. Here, we report that the off-target effects of both A > G and C > T editors can be dramatically reduced without compromising the on-target editing simply by inserting the editing enzymes into the middle of nCas9 at tolerant sites identified using a transposon-based genetic screen. Furthermore, employing this Cas-embedding strategy, we have created a highly specific editor capable of efficient C > T editing at methylated and GC-rich sequences.

Occurrence of blaTEM and blaROB in Haemophilus species Causing Respiratory Tract Infections.

BACKGROUND: Emergence of resistance to some antibiotics in Haemophilus influenzae, a respiratory pathogen is a cause of concern. The aim is to study the antibiotic susceptibility pattern of Haemophilus isolates from respiratory infections with reference to beta-lactam resistance. METHODS: This is a laboratory based prospective study done in the department of microbiology in a tertiary care center after institutional ethics committee clearance. Haemophilus influenzae isolates from respiratory tract specimens over a period of one year were subjected to antibiotic susceptibility tests. Beta-lactamase production was detected by nitrocefin disc. hpd gene, blaTEM and blaROB genes were detected by PCR. The data was analysed using SPSS 11.5 version. RESULTS: Of the 162 isolates, 89.5% were from sputum specimens. Ampicillin resistance was seen in 5 (3.09%) isolates. The ampicillin resistant strains were positive for beta-lactamase enzyme and blaTEM gene. BLNAR and isolates with blaROB gene were not found. CONCLUSION: In case of Haemophilus influenzae respiratory tract infection empirical treatment with amoxicillin clavulanate or third generation cephalosporin may be the drugs of choice in our geographic area.

Epidemiology of Haemophilus influenzae in the Republic of Ireland, 2010-2018.

The purpose of this study was to investigate H. influenzae epidemiology in the Republic of Ireland. We performed serotyping, multi-locus sequence typing (MLST) and susceptibility testing on H. influenzae isolates received by the Irish Meningitis and Sepsis Reference Laboratory from 2010 to 2018. Three hundred sixty-seven invasive and 41 non-invasive infection (NII) isolates were received. Invasive isolates were mostly recovered from paediatric (21%) and elderly (42%) populations. Invasive disease was more prevalent in females of childbearing age (72%) compared with males the same age (28%). Non-typeable H. influenzae (NTHi) predominated among invasive (83%) and NII (95%). Invasive Hib disease isolates were infrequent (4%, n = 15). Among invasive disease, Hif was the commonest encapsulated serotype (10%, n = 37), and the only encapsulated serotype detected in NII (5%, 2/41). The first PCR-confirmed serotypes d and a in Ireland were characterised among invasive disease in 2017 and 2018, respectively. MLST revealed a diverse NTHi population, while encapsulated serotypes were clonal. Sequence type (ST) 103 (n = 14) occurred exclusively in invasive NTHi disease. Ampicillin resistance (AmpR) was 18% among invasive isolates and 22% in NII. ß-Lactamase production was the main source of ampicillin resistance in invasive and NII isolates. We detected ß-lactamase negative ampicillin resistance (BLNAR) among invasive isolates. We report an NTHi fluoroquinolone-resistant clone: ST1524 among invasive (n = 2) and NII isolates (n = 2). The Hib vaccine has positively impacted on Hib disease in Ireland, given the low frequency of Hib. The dominance of NTHi, emergence of serotypes a and d and BLNAR suggest a changing H. influenzae epidemiology in Ireland.

Antibacterial and antibiofilm effects of flufenamic acid against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infections are one of the most serious surgery complications, and their prevention is of utmost importance. Flufenamic acid is a non-steroid anti-inflammatory drug approved for clinical use to relieve inflammation and pain in rheumatoid arthritis patients. In this study, we explored the antibacterial efficacy of flufenamic acid and the mechanisms underlying this effect. By using minimal inhibitory concentration (MIC), time-kill, resistance induction assays, and the antibiotic synergy test, we demonstrated that flufenamic acid inhibited the growth of methicillin-resistant staphylococci and did not induce resistance when it was used at the MIC. Furthermore, flufenamic acid acted synergistically with the beta-lactam antibiotic oxacillin and did not show significant toxicity toward mammalian cells. The biofilm inhibition assay revealed that flufenamic acid could prevent biofilm formation on medical implants and destroy the ultrastructure of the bacterial cell wall. RNA sequencing and quantitative RT-PCR indicated that flufenamic acid inhibited the expression of genes associated with peptidoglycan biosynthesis, beta-lactam resistance, quorum sensing, and biofilm formation. Furthermore, flufenamic acid efficiently ameliorated a local infection caused by MRSA in mice. In conclusion, flufenamic acid may be a potent therapeutic compound against MRSA infections and a promising candidate for antimicrobial coating of implants and surgical devices.

Resistencia a los β -lactámicos en enterococos / Resistance to β-lactams in enterococci

Enterococci are intrinsically resistant to several antimicrobial classes and show a great ability to acquire new mechanisms of resistance. Resistance to β-lactam antibiotics is a major concern because these drugs either alone or in combination are commonly used for the treatment of enterococcal infections. Ampicillin resistance, which is rare in Enterococcus faecium occurs in most of the hospital-associated Enterococcus faecium isolates. High-level resistance to ampicillin in E. faecium is mainly due to the enhanced production of PBP5 and/or by polymorphisms in the beta subunit of this protein. The dissemination of high-level ampicillin resistance can be the result of both clonal spread of strains with mutated pbp5 genes and resistance horizontal gene transfer.

Los enterococos son intrínsecamente resistentes a varias clases de antimicrobianos y presentan una gran capacidad para adquirir mecanismos de resistencia. La resistencia a los antibióticos p-lactámicos es preocupante porque estos fármacos solos o combinados se usan comúnmente para el tratamiento de las infecciones enterocócicas. La mayoría de los aislamientos hospitalarios de Enterococcus faecium presentan resistencia a la ampicilina, la cual es rara en Enterococcus faecalis. El alto nivel de resistencia a la ampicilina en E. faecium se debe principalmente a la hiperproducción de PBP5 y/o a polimorfismos en la subunidad beta de esta proteína. La propagación de esta resistencia puede deberse tanto a la diseminación clonal de cepas con genes pbp5 mutados como a la transferencia horizontal de genes.

Amino acid substitution mutations and mRNA expression levels of the pbp5 gene in clinical Enterococcus faecium isolates conferring high level ampicillin resistance.

In this study, clinical ampicillin-resistant Enterococcus faecium isolates with minimum inhibitory concentrations (MICs) for ampicillin in the ranges from 128 to ˃512 µg/mL (n = 17) and two ampicillin-susceptible isolates (MIC 1 µg/mL) were investigated. No ß-lactamase production was detected in these isolates. Alterations in the C-terminal part of pbp5 and levels of pbp5 mRNA expression were investigated by sequencing and quantitative real-time qRT-PCR, respectively. Sequencing analysis revealed five different pbp5 alleles (A to E) having differences in 18 amino acid positions spanning from residue 426 to 642. Allele A (V-462 â†’ A, H-470 â†’ Q, M-485 â†’ A, N-496 â†’ K, A-499 â†’ T, E-525 â†’ D, N-546 â†’ T, A-558 â†’ T, G-582 â†’ S, E-629 â†’ V, K-632 â†’ Q, and P-642 â†’ L) was the most frequent allele. The presence of just two susceptible isolates in allele E suggests a possible correlation between amino acid patterns and MIC, even if there is no discernible correlation with specific single amino acid differences. Also, these were the only isolates that showed much lower expression of class B penicillin-binding protein 5 (PBP5) compared to isolates with MIC of 128 or greater. Thus, ampicillin MICs were correlated with PBP5 expression.

Molecular Characterization of Salmonella enterica Serovar Enteritidis, Genetic Basis of Antimicrobial Drug Resistance and Plasmid Diversity in Ampicillin-Resistant Isolates.

Salmonella enterica serovar Enteritidis is the most common cause of human salmonellosis worldwide. In this study, all clinical isolates of Salmonella Enteritidis recovered between January 2008 and June 2014 in a Spanish region (491) were screened for antimicrobial drug resistance and the phage type (PT) was determined for a significant number (265). PT1, PT14b, PT56, PT6, PT4, and PT8 were the predominant PTs, accounting together for 82% of the isolates. A total of 38.3% of the isolates were susceptible to all antimicrobials tested, 46.4% and 6.1% isolates were resistant to nalidixic acid and ampicillin, respectively, and single isolates were resistant to two (ampicillin and nalidixic acid) or six (ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, and trimethoprim) agents. Nalidixic acid resistance was statistically associated with PT1 and PT14b (p < 0.05, 95% CI), and ampicillin resistance with PT6/PT6a (p < 0.05, 95% CI). All ampicillin-resistant isolates (30) carried a plasmid-encoded blaTEM-1. All except one harbored the virulence plasmid specific of Salmonella Enteritidis (IncFIIA + IncFIB; 28 isolates) or a blaTEM-1-positive variant herein (IncFIIA + IncFIB; 1 isolate). Five additional blaTEM-1 plasmids, of the ColE1, IncX, IncF, and IncI incompatibility groups, were identified. The IncI plasmid, found in the single multidrug-resistant isolate, carried the strAB and sul2 genes together with genes of the virulence plasmid, including the spv operon. The obtained results highlight the high diversity of blaTEM-1 plasmids conferring ampicillin resistance in Salmonella Enteritidis, and support clonal expansion as the main cause of nalidixic acid resistance in this serovar.

Detection of beta-lactamase-negative ampicillin resistance in Haemophilus influenzae in Belgium.

Haemophilus influenzae, a frequent colonizer of the respiratory tract, is the causative agent of several clinically important infections. In cases that require therapeutic intervention, laboratory susceptibility testing can detect beta-lactam antibiotic resistance and guide the best treatment course. In the absence of a beta-lactamase, beta-lactam resistance may be due to an altered form of the PBP3 protein, encoded by the ftsI gene. While these so-called beta-lactamase-negative ampicillin-resistant (BLNAR) strains are of serious clinical interest, identification in the clinical laboratory is not always straightforward. In the current study, the ftsI genes of a set of phenotypic BLNAR H. influenzae isolates taken from samples collected in the UZ Brussel hospital in Belgium were sequenced and re-tested at the National Reference Laboratory (NRC). Non-silent mutations in the ftsI gene were found in 100% of the isolates. Although 30% of the isolates were classified by the NRC as beta-lactamase-negative ampicillin-sensitive (BLNAS) strains based on the EUCAST guidelines on ampicillin minimal inhibitory concentration (MIC), all isolates showed MIC values ≥1 mg/L. These relatively high MIC values indicate a decreased susceptibility to ampicillin, and suggest that sequencing of the ftsI gene should be used as part of an antibiotic susceptibility testing (AST) algorithm in the clinical laboratory. This would allow clinicians to make better informed decisions regarding patient treatment.

Increasing Prevalence of Group III Penicillin-Binding Protein 3 Mutations Conferring High-Level Resistance to Beta-Lactams Among Nontypeable Haemophilus influenzae Isolates from Children in Korea.

This study investigated the roles of ß-lactamase and penicillin-binding protein 3 (PBP3) alterations in the development of recent antimicrobial resistance in nontypeable Haemophilus influenzae (NTHi) isolated from Korean children. Nasopharyngeal NTHi isolates from children at a tertiary children's hospital were tested for antimicrobial susceptibility using E-test. ß-lactamase production was screened by the paper disc test, and polymerase chain reaction amplification of blaTEM and blaROB-1 was performed. The ftsI gene was amplified to identify PBP3 alteration. Of the 53 NTHi isolates, 69.8% were ampicillin nonsusceptible. The nonsusceptibility rates for cefaclor were 81.1%, cefpodoxime 69.8%, and amoxicillin/clavulanate 32.1%. About 60.3% and 32.1% of the isolates were genetically ß-lactamase-nonproducing ampicillin-resistant (gBLNAR) and genetically ß-lactamase-producing amoxicillin/clavulanate-resistant (gBLPACR) strains, respectively. Group III amino acid substitutions comprised 65.6% of the gBLNAR strains and 70.6% of the gBLPACR strains. MIC50 for amoxicillin/clavulanate, cefaclor, cefuroxime, cefpodoxime, and cefixime were more than 2-80 times higher in the gBLNAR and gBLPACR strains compared with gBLPAR strains. Group III gBLNAR strains had significantly higher ampicillin, amoxicillin/clavulanate, cefpodoxime, and cefixime minimum inhibitory concentrations than group II strains. Group III gBLNAR and gBLPACR NTHi strains are highly prevalent in Korea, raising the alarm about increasing ß-lactam resistance in NTHi.

Multiclonal Expansion and High Prevalence of ß-Lactamase-Negative Haemophilus influenzae with High-Level Ampicillin Resistance in Japan and Susceptibility to Quinolones.

ß-Lactam-resistant Haemophilus influenzae is a clinical concern. A high prevalence (>40%) of ß-lactamase-negative high-level ampicillin-resistant H. influenzae (high-BLNAR) isolates in Japan has been reported. However, the reasons for the expansion are unknown. High-BLNAR strains possess an amino acid substitution, either Asn526Lys (group III) or Arg517His (group III-like) in addition to Ser385Thr, in penicillin-binding protein 3 (PBP3). To determine the current prevalence of high-BLNAR strains and the mechanisms behind their expansion in Japan, their prevalence, PBP3 types, multilocus sequence types, and susceptibilities to quinolones approved in Japan as alternatives were determined. Sixty percent of H. influenzae clinical isolates (62/104 isolates) were ß-lactamase-negative ampicillin-resistant H. influenzae (BLNAR) strains. Among BLNAR isolates, 92% (57/62 isolates) were high-BLNAR strains. Most isolates were classified as belonging to group III, which contained many genotypes (11 PBP3 types and 25 sequence types). These results indicated that the expansion of high-BLNAR isolates was multiclonal and such strains are still predominant in Japanese clinical settings. One high-BLNAR isolate harbored the novel amino acid substitution Asn526Met in addition to Ser385Thr in PBP3, suggesting a new group (group IV). No quinolone-resistant H. influenzae isolates were identified. The MICs for the quinolones (moxifloxacin, garenoxacin, and tosufloxacin) were similar to that for levofloxacin, whereas sitafloxacin exhibited a lower MIC. However, we obtained 4 H. influenzae isolates with decreased quinolone susceptibility with the amino acid substitution Ser84Leu in GyrA, and 3 of those isolates were high-BLNAR isolates. In summary, this study shows that multiclonal high-BLNAR strains predominate in a Japanese university hospital. Isolates remain sensitive to quinolones, but vigilance is required to prevent the development of fluoroquinolone resistance in high-BLNAR strains.

Analysis of Virulence Factors and Antimicrobial Resistance in Salmonella Using Molecular Techniques and Identification of Clonal Relationships Among the Strains.

A total of 50 Salmonella enterica strains were isolated from clinical samples from 2009 to 2012 and analyzed for the presence of virulence genes found in SPI-1, SPI-2, and plasmids. The distribution and frequency of the antimicrobial resistance genes and plasmids were revealed, and pulsed-field gel electrophoresis (PFGE) patterns were investigated. Five genes were identified from the seven strains with resistance or intermediate resistance to ampicillin: blaSHV-1 (present in six strains), qnrS1 (present in five strains), blaTEM-1 (present in three strains), blaCTX-M-1 (present in one strain), and qnrB1 (present in one strain). One trimethoprim-sulfamethoxazole-resistant strain was positive for sulI but negative for sulII. In addition, we detected TEM-1 and qnrS1 in one strain; SHV-1 and qnrS1 in two strains; TEM-1, SHV-1, CTX-M-1, and qnrS1 in one strain; TEM-1, SHV-1, and qnrB1 in one strain; and SHV-1 and sulI genes in one strain together. Plasmid-based replicon typing assay revealed that all 50 strains carried FIIS, 13 carried I1, 1 carried I2, 4 carried P, 1 carried A/C, and 4 carried X1 replicon. PFGE was used to type 46 of the 50 strains and classify them into 22 major groups, 33 pulsotypes, and 8 major clusters. All strains carried all the virulence genes of interest on both Salmonella Pathogenicity Islands 1 and 2 and plasmids suggested high potential for pathogenicity. All antimicrobial-resistant strains contained at least one of the resistance genes of interest, confirming a phenotype-genotype association in antimicrobial resistance.

Association mapping from sequencing reads using k-mers.

Genome wide association studies (GWAS) rely on microarrays, or more recently mapping of sequencing reads, to genotype individuals. The reliance on prior sequencing of a reference genome limits the scope of association studies, and also precludes mapping associations outside of the reference. We present an alignment free method for association studies of categorical phenotypes based on counting [Formula: see text]-mers in whole-genome sequencing reads, testing for associations directly between [Formula: see text]-mers and the trait of interest, and local assembly of the statistically significant [Formula: see text]-mers to identify sequence differences. An analysis of the 1000 genomes data show that sequences identified by our method largely agree with results obtained using the standard approach. However, unlike standard GWAS, our method identifies associations with structural variations and sites not present in the reference genome. We also demonstrate that population stratification can be inferred from [Formula: see text]-mers. Finally, application to an E.coli dataset on ampicillin resistance validates the approach.