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Background Haemophilus influenzae is an opportunistic bacterial pathogen that can cause severe respiratory tract and invasive infections. The emergence of β-lactamase-negative ampicillin-resistant (BLNAR) strains and unclear correlations between genotypic (i.e., gBLNAR) and phenotypic resistance are challenging empirical treatments and patient management. Thus, we sought to revisit molecular resistance mechanisms and to identify new resistance determinants of H. influenzae . Methods We performed a systematic meta-analysis of H. influenzae isolates ( n  = 291) to quantify the association of phenotypic ampicillin and cefotaxime resistance with previously defined resistance groups, i.e., specific substitution patterns of the penicillin binding protein PBP3, encoded by ftsI . Using phylogenomics and a genome-wide association study (GWAS), we investigated evolutionary trajectories and novel resistance determinants in a public global cohort ( n  = 555) and a new clinical cohort from three European centers ( n  = 298), respectively. Results Our meta-analysis confirmed that PBP3 group II- and group III-related isolates were significantly associated with phenotypic resistance to ampicillin ( p  < 0.001), while only group III-related isolates were associated with resistance to cefotaxime ( p  = 0.02). The vast majority of H. influenzae isolates not classified into a PBP3 resistance group were ampicillin and cefotaxime susceptible. However, particularly group II isolates had low specificities (< 16%) to rule in ampicillin resistance due to clinical breakpoints classifying many of them as phenotypically susceptible. We found indications for positive selection of multiple PBP3 substitutions, which evolved independently and often step-wise in different phylogenetic clades. Beyond ftsI , other possible candidate genes (e.g., oppA, ridA, and ompP2 ) were moderately associated with ampicillin resistance in the GWAS. The PBP3 substitutions M377I, A502V, N526K, V547I, and N569S were most strongly related to ampicillin resistance and occurred in combination in the most prevalent resistant haplotype H1 in our clinical cohort. Conclusions Gradient agar diffusion strips and broth microdilution assays do not consistently classify isolates from PBP3 groups as phenotypically resistant . Consequently, when the minimum inhibitory concentration is close to the clinical breakpoints, and genotypic data is available, PBP3 resistance groups should be prioritized over susceptible phenotypic results for ampicillin. The implications on treatment outcome and bacterial fitness of other extended PBP3 substitution patterns and novel candidate genes need to be determined.

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. β-Lactamase production was the main source of ampicillin resistance in invasive and NII isolates. We detected β-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.

As the most consumed shrimp variety, white-leg shrimp ( Litopenaeus vannamei ) owns a high market demand in Korea. This study sought to screen the frozen white-leg shrimp for Aeromonas spp. harboring antimicrobial and heavy metal resistance characteristics. A total of 44 Aeromonas spp. strains were isolated and tested for antibiotic susceptibility and heavy metal tolerance followed by PCR-based detection of resistance genes and integrons. It was observed that resistance to ampicillin and oxacillin was 100% among isolates. Besides, 95%, 89%, 86%, 80%, 66%, and 43% of the isolates were resistant to nalidixic acid, tetracycline, cephalothin, streptomycin, trimethoprim–sulfamethoxazole, and imipenem, respectively, and less resistance to other antibiotics was also observed. Cr resistance was the highest (91%) among five heavy metals tested, whereas 57%, 32%, 20%, and 9% of the isolates were tolerant to Cu, Pb, Cd, and Hg, respectively. The PCR assays implied the presence of qnrB, qnrS, tetA, tetE, aac(6 ′ )-Ib, and aphAI-IAB, and intI1 genes among 80%, 77%, 18%, 30%, 9%, 0.25%, and 82% of the isolates, respectively. There were 35 (80%) integron 1-positive isolates harboring qacE2, dfrA1, orfC, orfD, aadB, catB3, oxa-10, and aadA1 genes in varying combinations. In addition, heavy metal resistance genes, CopA , merA , and CzcA were positive in 9%, 7%, and 27% of the isolates, respectively. According to these outcomes, the frozen white-leg shrimp in Korean markets can be suggested as a source of multidrug and heavy metal-resistant Aeromonas spp. that carries genetic determinants.

is commonly isolated from the airways of COPD patients. Antibiotic treatment may cause the emergence of resistant strains, particularly ampicillin-resistant strains, including β-lactamase-negative ampicillin resistance (BLNAR) strains. Genetic identification using sequencing is the optimum method for identifying mutations within BLNAR strains. The prevalence of BLNAR in COPD patients during the stable state has not been reported. We investigated the antibiotic resistance patterns of present in the sputum of stable COPD patients, focusing on ampicillin resistance; the prevalence of enzyme and non-enzyme-mediated ampicillin resistance was determined. A subset of patients was followed up longitudinally to study strain switching and antibiotic sensitivity changes. Sputum sampling was performed in 61 COPD patients, with 42 samples obtained at baseline; was detected by polymerase chain reaction in 28 samples. In all, 45 patients completed the follow-up for 2 years; 24 isolates were obtained. Disk diffusion showed the highest antibiotic resistance in the penicillin antibiotic group (eg, 67% for ampicillin) and macrolides (eg, 46% for erythromycin), whereas all isolates were susceptible to quinolones. Of the 16 isolates resistant to ampicillin, 9 (56%) were β-lactamase positive. The β-lactamase-negative isolates were further investigated; none of these fulfilled the phenotypic BLNAR classification criteria of ampicillin minimum inhibitory concentration >1 µg/mL, and only one demonstrated an mutation. Frequent strain switching was confirmed using multilocus sequence typing and was associated with changes in the antibiotic sensitivity pattern. We observed an overidentification of ampicillin resistance by disk diffusion. The majority of ampicillin resistance was due to enzyme production. strain changes during the stable state may be associated with a change in antibiotic sensitivity; this has implications for empirical antibiotic prescribing.

Background: The Enterococcus faecium genogroup, referred to as clonal complex 17 (CC17), seems to possess multiple determinants that increase its ability to survive and cause disease in nosocomial environments. Methods: Using 53 clinical and geographically diverse US E. faecium isolates dating from 1971 to 1994, we determined the multilocus sequence type; the presence of 16 putative virulence genes (hyl Efm, esp Efm, and fms genes); resistance to ampicillin (AMP) and vancomycin (VAN); and high-level resistance to gentamicin and streptomycin. Results: Overall, 16 different sequence types (STs), mostly CC17 isolates, were identified in 9 different regions of the United States. The earliest CC17 isolates were part of an outbreak that occurred in 1982 in Richmond, Virginia. The characteristics of CC17 isolates included increases in resistance to AMP, the presence of hyl Efm and esp Efm, emergence of resistance to VAN, and the presence of at least 13 of 14 fms genes. Eight of 41 of the early isolates with resistance to AMP, however, were not in CC17. Conclusions: Although not all early US AMP isolates were clonally related, E. faecium CC17 isolates have been circulating in the United States since at least 1982 and appear to have progressively acquired additional virulence and antibiotic resistance determinants, perhaps explaining the recent success of this species in the hospital environment.

To identify the predictive and prognostic factors associated with ampicillin-resistant enterococcal bacteraemia, we retrospectively reviewed demographic, microbiological and clinical data of patients attending the Kyoto University Hospital, Japan, between 2009 and 2015. Logistic regression and Cox regression analyses were performed to determine the predictive and prognostic factors, respectively. In total, 235 episodes of enterococcal bacteraemia were identified. As ampicillin susceptibility was uniform for Enterococcus faecalis isolates and almost all ampicillin-resistant isolates were E. faecium , bacteraemia due to these species was investigated separately. E. faecalis and E. faecium accounted for 41.7% (98/235) and 48.1% (113/235) of the isolates, respectively and 91.2% of all E. faecium were ampicillin resistant. Nosocomial E. faecium bacteraemia acquisition (odds ratio (OR), 13.6; 95% confidence intervals, 3.16–58.3) was associated with ampicillin-resistant isolates. Bacteraemia from an unknown source (hazard ratio (HR), 2.91; 95% CI 1.36–6.21) and an increased Pitt bacteraemia score (PBS) (HR, 1.36; 95% CI 1.21–1.52) were associated with 30-day mortality in E. faecium infections. Likewise, bacteraemia from an unknown source (HR, 4.17; 95% CI 1.25–13.9) and increased PBS (HR, 1.27; 95% CI 1.09–1.48) were associated with 30-day mortality in patients with E. faecalis bacteraemia. The empirical therapeutic administration of glycopeptides is recommended for patients with bacteraemia from an unknown source in whom severe E. faecium bacteraemia is suspected.

Background The widespread administration of the Haemophilus influenzae type b vaccine has led to the predominance of non-typable H. influenzae (NTHi). However, the occurrence of invasive NTHi infection based on gynecologic diseases is still rare. Case presentation A 51-year-old Japanese woman with a history of adenomyoma presented with fever. Blood cultures and a vaginal discharge culture were positive with NTHi. With the high uptake in the uterus with 67 Ga scintigraphy, she was diagnosed with invasive NTHi infection. In addition to antibiotic administrations, a total hysterectomy was performed. The pathological analysis found microabscess formations in adenomyosis. Conclusions Although NTHi bacteremia consequent to a microabscess in adenomyosis is rare, this case emphasizes the need to consider the uterus as a potential source of infection in patients with underlying gynecological diseases, including an invasive NTHi infection with no known primary focus.

Eighty-two nonduplicated ampicillin-resistant Enterococcus faecium (AREF) isolates from clinical infections at the Charles Nicolle Hospital of Tunisia were investigated. They were collected from January 2001 to December 2009. Genetic relationship between them was studied using pulsed-field gel electrophoresis. The amino acid sequence difference variations of the C-terminal part of penicillin-binding protein 5 (PBP5) versus levels of expressed mRNA were investigated by polymerase chain reaction (PCR), sequencing, and real-time PCR quantification of (PBP5), respectively. No β-lactamase activity was detected and none of our strains showed resistance to glycopeptides, which retain their therapeutic efficiency against enterococcal infections in our hospital. Pattern analysis of the strains revealed six main clones disseminating in different wards. Sequence data revealed the existence of 19 different plp5 alleles with a difference in 16 amino acid positions spanning from residue 414 to 632. Each allele presented at least five amino acid substitutions (His-470→Gln, Asn-496→Lys, Ala-499→Thr, Glu-525→Asp, and Glu-629→Val). No correlation between amino acid sequence polymorphism of PBP5 and levels of ampicillin resistance was detected. The levels of plp5 mRNA expression varied between strains and did not always correlate with levels of ampicillin resistance in clinical AREF.

Ampicillin and vancomycin are important antibiotics for the therapy of Enterococcus faecalis infections. The ampicillin resistance gene pbp5 is intrinsic in Enterococcus faecium . The vanC1 gene confers resistance to vancomycin and serves as a species marker for Enterococcus gallinarum . Both genes are chromosomally located. Resistance to ampicillin and vancomycin was determined in 484 E. faecali s of human and porcine origin by microdilution. Since E. faecalis are highly skilled to acquire resistance genes, all strains were investigated for the presence of pbp5 (and, in positive strains, for the penicillin-binding protein synthesis repressor gene psr ) and vanC1 (and, in positive strains, for vanXYc and vanT ) by using polymerase chain reaction (PCR). One porcine and one human isolate were phenotypically resistant to ampicillin; no strain was vancomycin resistant. Four E. faecalis (3/1 of porcine/human origin) carried pbp5 (MIC=1 mg/L), and four porcine strains were vanC1 positive (minimum inhibitory concentration [MIC]=1 mg/L). Real-time reverse transcriptase (RT)-PCR revealed that the genes were not expressed. The psr gene was absent in the four pbp5 -positive strains; the vanXYc gene was absent in the four vanC1 -positive strains. However, vanT of the vanC gene cluster was detected in two vanC1 -positive strains. To our knowledge, this is the first report on the presence of pbp5 , identical with the “ E. faecium pbp5 gene,” and of vanC1 / vanT in E. faecalis . Even if resistance is not expressed in these strains, this study shows that E. faecalis have a strong ability to acquire resistance genes—and potentially to spread them to other bacteria. Therefore, close monitoring of this species should be continued.

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.