Geographical migration and fitness dynamics of Streptococcus pneumoniae.

Streptococcus pneumoniae is a leading cause of pneumonia and meningitis worldwide. Many different serotypes co-circulate endemically in any one location1,2. The extent and mechanisms of spread and vaccine-driven changes in fitness and antimicrobial resistance remain largely unquantified. Here using geolocated genome sequences from South Africa (n = 6,910, collected from 2000 to 2014), we developed models to reconstruct spread, pairing detailed human mobility data and genomic data. Separately, we estimated the population-level changes in fitness of strains that are included (vaccine type (VT)) and not included (non-vaccine type (NVT)) in pneumococcal conjugate vaccines, first implemented in South Africa in 2009. Differences in strain fitness between those that are and are not resistant to penicillin were also evaluated. We found that pneumococci only become homogenously mixed across South Africa after 50 years of transmission, with the slow spread driven by the focal nature of human mobility. Furthermore, in the years following vaccine implementation, the relative fitness of NVT compared with VT strains increased (relative risk of 1.68; 95% confidence interval of 1.59-1.77), with an increasing proportion of these NVT strains becoming resistant to penicillin. Our findings point to highly entrenched, slow transmission and indicate that initial vaccine-linked decreases in antimicrobial resistance may be transient.

Loss of Pde1 function acts as an evolutionary gateway to penicillin resistance in Streptococcus pneumoniae.

Streptococcus pneumoniae is a major human pathogen and rising resistance to ß-lactam antibiotics, such as penicillin, is a significant threat to global public health. Mutations occurring in the penicillin-binding proteins (PBPs) can confer high-level penicillin resistance but other poorly understood genetic factors are also important. Here, we combined strictly controlled laboratory experiments and population analyses to identify a new penicillin resistance pathway that is independent of PBP modification. Initial laboratory selection experiments identified high-frequency pde1 mutations conferring S. pneumoniae penicillin resistance. The importance of variation at the pde1 locus was confirmed in natural and clinical populations in an analysis of >7,200 S. pneumoniae genomes. The pde1 mutations identified by these approaches reduce the hydrolytic activity of the Pde1 enzyme in bacterial cells and thereby elevate levels of cyclic-di-adenosine monophosphate and penicillin resistance. Our results reveal rapid de novo loss of function mutations in pde1 as an evolutionary gateway conferring low-level penicillin resistance. This relatively simple genomic change allows cells to persist in populations on an adaptive evolutionary pathway to acquire further genetic changes and high-level penicillin resistance.

Streptococcus suis serotype 9 in Italy: genomic insights into high-risk clones with emerging resistance to penicillin.

BACKGROUND: Streptococcus suis is an important pig pathogen and an emerging zoonotic agent. In a previous study, we described a high proportion of penicillin-resistant serotype 9 S. suis (SS9) isolates on pig farms in Italy. OBJECTIVES: We hypothesized that resistance to penicillin emerged in some SS9 lineages characterized by substitutions at the PBPs, contributing to the successful spread of these lineages in the last 20 years. METHODS: Sixty-six SS9 isolates from cases of streptococcosis in pigs were investigated for susceptibility to penicillin, ceftiofur and ampicillin. The isolates were characterized for ST, virulence profile, and antimicrobial resistance genes through WGS. Multiple linear regression models were employed to investigate the associations between STs, year of isolation, substitutions at the PBPs and an increase in MIC values to ß-lactams. RESULTS: MIC values to penicillin increased by 4% each year in the study period. Higher MIC values for penicillin were also positively associated with ST123, ST1540 and ST1953 compared with ST16. The PBP sequences presented a mosaic organization of blocks. Within the same ST, substitutions at the PBPs were generally more frequent in recent isolates. Resistance to penicillin was driven by substitutions at PBP2b, including K479T, D512E and K513E, and PBP2x, including T551S, while reduced susceptibility to ceftiofur and ampicillin were largely dependent on substitutions at PBP2x. CONCLUSIONS: Here, we identify the STs and substitutions at the PBPs responsible for increased resistance of SS9 to penicillin on Italian pig farms. Our data highlight the need for monitoring the evolution of S. suis in the coming years.

The acquisition of clinically relevant amoxicillin resistance in Streptococcus pneumoniae requires ordered horizontal gene transfer of four loci.

Understanding how antimicrobial resistance spreads is critical for optimal application of new treatments. In the naturally competent human pathogen Streptococcus pneumoniae, resistance to ß-lactam antibiotics is mediated by recombination events in genes encoding the target proteins, resulting in reduced drug binding affinity. However, for the front-line antibiotic amoxicillin, the exact mechanism of resistance still needs to be elucidated. Through successive rounds of transformation with genomic DNA from a clinically resistant isolate, we followed amoxicillin resistance development. Using whole genome sequencing, we showed that multiple recombination events occurred at different loci during one round of transformation. We found examples of non-contiguous recombination, and demonstrated that this could occur either through multiple D-loop formation from one donor DNA molecule, or by the integration of multiple DNA fragments. We also show that the final minimum inhibitory concentration (MIC) differs depending on recipient genome, explained by differences in the extent of recombination at key loci. Finally, through back transformations of mutant alleles and fluorescently labelled penicillin (bocillin-FL) binding assays, we confirm that pbp1a, pbp2b, pbp2x, and murM are the main resistance determinants for amoxicillin resistance, and that the order of allele uptake is important for successful resistance evolution. We conclude that recombination events are complex, and that this complexity contributes to the highly diverse genotypes of amoxicillin-resistant pneumococcal isolates.

Penicillin and Cefotaxime Resistance of Quinolone-Resistant Neisseria meningitidis Clonal Complex 4821, Shanghai, China, 1965-2020.

Clonal complex 4821 (CC4821) Neisseria meningitidis, usually resistant to quinolones but susceptible to penicillin and third-generation cephalosporins, is increasing worldwide. To characterize the penicillin-nonsusceptible (PenNS) meningococci, we analyzed 491 meningococci and 724 commensal Neisseria isolates in Shanghai, China, during 1965-2020. The PenNS proportion increased from 0.3% in 1965-1985 to 7.0% in 2005-2014 and to 33.3% in 2015-2020. Of the 26 PenNS meningococci, 11 (42.3%) belonged to the CC4821 cluster; all possessed mutations in penicillin-binding protein 2, mostly from commensal Neisseria. Genetic analyses and transformation identified potential donors of 6 penA alleles. Three PenNS meningococci were resistant to cefotaxime, 2 within the CC4821 cluster. With 96% of the PenNS meningococci beyond the coverage of scheduled vaccination and the cefotaxime-resistant isolates all from toddlers, quinolone-resistant CC4821 has acquired penicillin and cefotaxime resistance closely related to the internationally disseminated ceftriaxone-resistant gonococcal FC428 clone, posing a greater threat especially to young children.

Penicillin resistance in bovine Staphylococcus aureus: Genomic evaluation of the discrepancy between phenotypic and molecular test methods.

Staphylococcus aureus is a major pathogen in humans and animals. In cattle, it is one of the most important agents of mastitis, causing serious costs in the dairy industry. Early diagnosis and adequate therapy are therefore 2 key factors to deal with the problems caused by this bacterium, and benzylpenicillin (penicillin) is usually the first choice to treat these infections. Unfortunately, penicillin resistance testing in bovine S. aureus strains shows discrepant results depending on the test used; consequently, the best method for assessing penicillin resistance is still unknown. The aim of this study was therefore to find a method that assesses penicillin resistance in S. aureus and to elucidate the mechanisms leading to the observed discrepancies. A total of 146 methicillin-sensitive S. aureus strains isolated from bovine mastitis were tested for penicillin resistance using a broth microdilution [minimum inhibitory concentration (MIC)] and 2 different disk diffusion protocols. Furthermore, the strains were analyzed for the presence of the bla operon genes (blaI, blaR1, blaZ) by PCR, and a subset of 45 strains was also subjected to whole genome sequencing (WGS). Discrepant results were obtained when penicillin resistance of bovine S. aureus was evaluated by disk diffusion, MIC, and PCR methods. The discrepancies, however, could be fully explained by WGS analysis. In fact, it turned out that penicillin resistance is highly dependent on the completeness of the bla operon promotor: when the bla operon was complete based on WGS analysis, all strains showed MIC ≥1 µg/mL, whereas when the bla operon was mutated (31-nucleotide deletion), they were penicillin sensitive except in those strains where an additional, bla operon-independent resistance mechanism was observed. Further, WGS analyses showed that penicillin resistance is truly assessed by the MIC assay. In contrast, caution is required when interpreting disk diffusion and PCR results.

Serogroup Y Clonal Complex 23 Meningococcus in China Acquiring Penicillin Resistance from Commensal Neisseria lactamica Species.

Invasive meningococcal disease (IMD) due to serogroup Y Neisseria meningitidis (NmY) is rare in China; recently, an invasive NmY isolate, Nm512, was discovered in Shanghai with decreased susceptibility to penicillin (PenNS). Here, we investigated the epidemiology of NmY isolates in Shanghai and explored the potential commensal Neisseria lactamica donor of the PenNS NmY isolate. A total of 491 N. meningitidis and 724 commensal Neisseria spp. isolates were collected. Eleven NmY isolates were discovered from IMD (n = 1) and carriers (n = 10), including two PenNS isolates with five-key-mutation-harboring (F504L-A510V-I515V-H541N-I566V) penA genes. Five of the eight ST-175 complex (CC175) isolates had a genotype [Y:P1.5-1,2-2:F5-8:ST-175(CC175)] identical to that of the predominant invasive clone found in South Africa. Only one invasive NmY CC23 isolate (Nm512) was discovered; this isolate carried a novel PenNSpenA832 allele, which was identified in commensal N. lactamica isolates locally. Recombination analysis and transformation of the penA allele highlighted that N. meningitidis Nm512 may acquire resistance from its commensal donor; this was supported by the similar distribution of transformation-required DNA uptake sequence variants and the highly cognate receptor ComP between N. meningitidis and N. lactamica. In 2,309 NmY CC23 genomes from the PubMLST database, isolates with key-mutation-harboring penA genes comprised 12% and have been increasing since the 1990s, accompanied by recruitment of the blaROB-1 and/or quinolone resistance allele. Moreover, penA22 was predominant among genomes without key mutations in penA. These results strongly suggest that Nm512 is a descendant of the penA22-harboring CC23 isolate from Europe and acquired its penicillin resistance locally from commensal N. lactamica species by natural transformation.

Penicillin- and Cephalosporin-Resistant Pneumococcal Meningitis: Treatment in the Real World and in Guidelines.

To report on the therapy used for penicillin- and cephalosporin-resistant pneumococcal meningitis, we conducted an observational cohort study of patients admitted to our hospital with pneumococcal meningitis between 1977 and 2018. According to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations, we defined pneumococci as susceptible and resistant to penicillin with MIC values of ≤0.06 mg/L and > 0.06 mg/L, respectively; the corresponding values for cefotaxime (CTX) were ≤0.5 mg/L and >0.5 mg/L. We treated 363 episodes of pneumococcal meningitis during the study period. Of these, 24 had no viable strain, leaving 339 episodes with a known MIC for inclusion. Penicillin-susceptible strains accounted for 246 episodes (73%), penicillin-resistant strains for 93 (27%), CTX susceptible for 58, and CTX resistant for 35. Nine patients failed or relapsed and 69 died (20%), of whom 22% were among susceptible cases and 17% were among resistant cases. During the dexamethasone period, mortality was equal (12%) in both susceptible and resistant cases. High-dose CTX (300 mg/Kg/day) helped to treat failed or relapsed cases and protected against failure when used as empirical therapy (P = 0.02), even in CTX-resistant cases. High-dose CTX is a good empirical therapy option for pneumococcal meningitis in the presence of a high prevalence of penicillin and cephalosporin resistance, effectively treating pneumococcal strains with MICs up to 2 mg/L for either penicillin or CTX.

Screening for ß-lactam resistance by penicillin G in the Streptococcus anginosus group challenged by rare strains with altered PBPs.

BACKGROUND: Streptococcus anginosus group (SAG) strains are common pathogens causing abscesses and bacteraemia. They are generally susceptible to ß-lactams, which constitute first-line treatment. EUCAST recommends testing penicillin G susceptibility to screen for ß-lactam resistance. Isolates categorized as susceptible (negative screening) can be reported as susceptible to aminopenicillins and third-generation cephalosporins. OBJECTIVES: To assess the reliability of penicillin G resistance screening in predicting ß-lactam resistance in SAG blood culture isolates, and to investigate isolates for which this test would be unreliable. METHODS: We determined the susceptibility to penicillin G, amoxicillin and ceftriaxone of 90 SAG blood culture isolates, all with negative penicillin G resistance screening. ß-Lactam-resistant strains were sequenced and compared with susceptible reference SAG strains. RESULTS: We detected two isolates displaying ß-lactam resistance, especially to third-generation cephalosporins, despite negative screening for penicillin G resistance. For these isolates, amino acid substitutions were identified next to the essential PBP motifs SxxK, SxN and/or KS/TGS/T. Changes in these motifs have been previously linked to ß-lactam resistance in Streptococcus pneumoniae. CONCLUSIONS: Our study suggests that aminopenicillin and third-generation cephalosporin susceptibility should be determined for SAG strains in the event of severe infection as screening for penicillin G resistance might not be sufficient to detect resistance mechanisms that predominantly affect cephalosporins. The PBP sequencing of resistant SAG strains allowed us to detect amino acid changes potentially linked to ß-lactam resistance.

Clinical and bacterial features of Group B streptococci with reduced penicillin susceptibility from respiratory specimens: a case-control study.

Streptococcus agalactiae (Group B Streptococcus, GBS) is an invasive pathogen that causes sepsis and meningitis among infants, elderly adults, and immunosuppressed patients. Generally, GBS is susceptible to penicillin; however, GBS with reduced penicillin susceptibility (PRGBS) has been reported. PRGBS are commonly isolated from respiratory specimens, but clinical features of patients with PRGBS remain unclear. In this case-control study, clinical features of patients with PRGBS and bacterial characteristics of these isolates from respiratory specimens were investigated. Patients with GBS at the University of the Ryukyus Hospital between January 2017 and June 2018 were retrospectively investigated. GBS were further classified into penicillin-susceptible GBS (PSGBS) and PRGBS using a drug susceptibility test. Moreover, serotypes, genotypes, and drug resistance genes of PRGBS isolates were determined. In total, 362 GBS were isolated, of which 46 were collected from respiratory specimens, which had the highest rate of PRGBS (24%). Compared to patients with PSGBS, those with PRGBS were more likely to have neuromuscular disease, poor performance status, risk of multidrug-resistant pathogen infection, prior pneumonia history within 1 year, and prior penicillin use within 1 year. Among eight PRGBS isolates, multilocus sequence typing revealed that five isolates were sequence type (ST) 358, two were ST3 and ST10, respectively, and one isolate was ST1404. All PRGBS isolates belonged to the ST1/ST19/ST10 group. This study reveals clinical characteristics of patients with PRGBS from respiratory specimens. Because invasive GBS infection cases are increasing, especially in the elderly, more attention should be paid to this infection.

Possible misinterpretation of penicillin susceptibility in Staphylococcus aureus blood isolate due to in vitro loss of the blaZ gene.

We describe a case of recurrent catheter-related blood stream infections (BSI) with Staphylococcus aureus, in which the first isolate tested susceptible to penicillin, while subsequent isolates were resistant. Phenotypic susceptibility correlated with the absence/presence of the blaZ gene. The in vitro stability of penicillin resistance was investigated by subculturing single colonies. In two out of five colonies, phenotypical resistance was lost after a single subculture, which correlated with loss of the blaZ gene. This in vitro phenomenon probably resulted in a very major error in the microbiology report of the first BSI, where penicillin had been recommended as treatment.

Diversity of amino acid substitutions of penicillin-binding proteins in penicillin-non-susceptible and non-vaccine type Streptococcus pneumoniae.

PURPOSE: In Japan, the introduction of pneumococcal conjugate vaccine (PCV) in children has decreased vaccine-type (VT) pneumococcal infections caused by penicillin (PEN)-non-susceptible Streptococcus pneumoniae. PEN-non-susceptible strains have gradually emerged among non-vaccine types (NVT). In this study, we aim to investigate the pbp gene mutations and the characteristics of PEN-binding proteins (PBPs) that mediate PEN resistance in NVT strains. MATERIALS AND METHODS: Pneumococcal 41 strains of NVT isolated from patients with invasive pneumococcal infection were randomly selected. Nucleotide sequences for pbp genes encoding PBP1A, PBP2X, and PBP2B were analyzed, and amino acid (AA) substitutions that contribute to ß-lactam resistance were identified. In addition, the three-dimensional (3D) structure of abnormal PBPs in the resistant strain was compared with that of a reference R6 strain via homology modeling. RESULTS: In PEN-non-susceptible NVT strains, Thr to Ala or Ser substitutions in the conserved AA motif (STMK) were important in PBP1A and PBP2X. In PBP2B, substitutions from Thr to Ala, adjacent to the SSN motif, and from Glu to Gly were essential. The 3D structure modeling indicated that AA substitutions are characterized by accumulation around the enzymatic active pocket in PBPs. Many AA substitutions detected throughout the PBP domains were not associated with resistance, except for AA substitutions in or adjacent to AA motifs. Clonal complexes and sequence types showed that almost all NVT cases originated in other countries and spread to Japan via repeat mutations. CONCLUSIONS: NVT with diverse AA substitutions increased gradually with pressure from both antimicrobial agents and vaccines.

Penicillin- and third-generation cephalosporin-resistant strains of Streptococcus pneumoniae meningitis: Case report and literature review.

BACKGROUND: Treatment of patients with penicillin-resistant S. pneumoniae (PRSP) is complicated because of the relatively poor blood-brain barrier penetration of effective antimicrobials. Our case: A previously healthy 70-year-old woman, a traveler from China to Japan, was admitted to our hospital with fever and loss of consciousness. She has no history of pneumococcal vaccination. She was diagnosed with bacterial meningitis due to penicillin-and third-generation cephalosporin-resistant strains of S. pneumoniae. The patient was successfully treated with a combination therapy of vancomycin (VCM) and levofloxacin (LVFX) and recovered without any neurological sequelae. As the treatment of penicillin-and third-generation cephalosporin-resistant strains of S. pneumoniae meningitis remains unclear, we conducted a review of the reported cases of meningitis caused by penicillin- and cephalosporin-resistant S. pneumoniae. METHOD: We performed a search using the keywords "penicillin-resistant Streptococcus pneumoniae," "meningitis," and "pneumococcal meningitis". We searched the electronic databases PubMed, Embase, and Ichushi from their inception to March 2020. Subsequently, two authors independently reviewed the resulting database records, retrieved full texts for eligibility assessment, and extracted data from these cases. RESULT: We identified 18 papers describing thirty-five cases of penicillin- and cephalosporin-resistant S. pneumoniae meningitis including our case. The patient's characteristics were; median age: 50 years, men:50%, 85% of cases received combination regimens of antibiotics: Ceftroriaxone (CTRX) plus VCM (20 cases), CTRX plus VCM plus rifampicin (RFP) (two cases), CTRX plus linezolid (one case), fluoroquinolones (two cases), carbapenems (six cases), Thirty-five percent received steroids. Twenty-four percent of patients died. Twenty-six percent of patients complicated neurological sequalae. CONCLUSION: Combination therapy including VCM plus LVFX could be a treatment option.

The impact of pneumococcal conjugate vaccine-13 on the incidence of pediatric community-acquired bacteremia.

The purpose of this study was to estimate the impact of pneumococcal conjugate vaccine-13 (PCV-13) introduction into the national immunization program in Israel on pneumococcal and non-pneumococcal pediatric community-acquired bacteremia (CAB). This is a retrospective cohort study, including children ≤ 18 years old with CAB, who were hospitalized in Rambam Health Care Campus, a tertiary medical center serving northern Israel, between the years 2004 and 2016. The proportional admission rate of pneumococcal bacteremia among all CAB events and the incidence of CAB and pneumococcal bacteremia per 1000 hospital admissions were compared between the pre- and post-pneumococcal vaccine eras. A total of 275 CAB events were identified. Common isolates were Streptococcus pneumoniae (SPn) (26.9%), Staphylococcus aureus (12.4%), Brucella spp. (11.6%), E. coli (10.9%), and Streptococcus pyogenes (5.8%). The pneumococcal bacteremia rate per 1000 hospital admissions decreased significantly from 1.59 to 0.6 (p < 0.001). The proportional pneumococcal bacteremia rate decreased from 55 (34.4%) to 19 (16.5%) (p 0.001). Penicillin resistance among pneumococcal isolates decreased dramatically from 50.9 to 5.3% (p < 0.001). The rate of bacteremia caused by other pathogens has not been changed significantly at the post-vaccination era (p 0.053). However, an increase in the incidence of S. pyogenes bacteremia from 1.9 to 11.3% (p < 0.001) was noticed. In addition, an outbreak of Brucella bacteremia occurred during the years 2015-2016. This study demonstrates the double positive effect of PVC-13 introduction: a sharp decrease in the proportional rate of pneumococcal bacteremia and in the resistance of SPn to penicillin. Also, there was a moderate decline in the incidence of CAB in exception to bacteremia caused by S. pyogenes. This trend was reversed due to a Brucella outbreak.

Comparative genomics of invasive Streptococcus pneumoniae CC320/271 serotype 19F/19A before the introduction of pneumococcal vaccine in India.

The emergence of multi drug resistant clone CC320 serotype19F/19A and their capsular (cps) antigenic variants due to selective pressures such as vaccine had been reported worldwide. Hence, it is important to identify the prevalent clones, sequence types and cps variants of serotype 19F/19A in India, where PCV13 has been recently introduced. Multi-locus sequence typing (MLST) was performed for all (n = 21) invasive S. pneumoniae isolates of serotype 19A (n = 5) and 19F (n = 16) collected between the years 2012 and 2018 from children less than 5 years. The genome characterization by whole genome sequencing for the Sequence types (STs) 320 and 271(n = 7) were performed and compared with another six Indian WGSs of similar STs available from the GPS platform. The predominant STs in the serotype 19F/19A study isolates were of CC320: ST 320, 236 and 271, associated with PMEN clone Taiwan19F-14. The WGSs of CC320 study isolates showed high genomic similarity to the Taiwan19F-14 clone, and the penicillin binding protein (PBP) amino acid sequence similarity was 100% for PBP1A, 93% for PBP 2B and 2X. Whilst PBP comparison with other global MDR ST320 strains revealed that the ST320 clones in India are of low-level penicillin resistance. The presence of a few ST320/19A/19F invasive isolates with high similarity to the Taiwan clone suggests slow and gradual expansion of Taiwan19F-14 associated CC320 clones in India. Since serotype 19F/19A is covered by PCV13 vaccine, the expansion of 19F/19A cones with non-PCV13 vaccine serotype in India should be monitored.

Occurrence and aetiology of subclinical mastitis in water buffalo in Bangladesh.

Subclinical mastitis (SCM) in water buffalo is a production disease associated with decreased milk yield and impaired milk quality and safety. Water buffalo is an important livestock species in Bangladesh, but information about the occurrence and aetiology of SCM in this species is scarce. A cross-sectional study was conducted as part of the Udder Health Bangladesh Programme to (i) determine the occurrence of SCM and bulk milk somatic cell count (SCC) in water buffalo in Bangladesh, (ii) identify pathogens causing SCM and (iii) evaluate penicillin resistance in isolated staphylococci strains. Sixteen buffalo farms in the Bagerhat and Noakhali regions of Bangladesh were selected for study and a bulk milk sample was collected from each farm. In addition, 299 udder quarter milk samples were collected from 76 animals. The bulk milk samples were assessed by direct SCC and the quarter milk samples by California mastitis test (CMT). The occurrence of SCM calculated at quarter and animal level was 42.5 and 81.6%, respectively. Milk samples from 108 CMT-positive quarters in 48 animals and 38 randomly selected CMT-negative quarters in 24 animals were investigated using bacteriological culture. Estimated mean bulk milk SCC was 195 000 cells/ml milk (range 47 000- 587 000 cells/ml milk). On culture, estimated quarter-level intramammary infection (IMI) was 40.4%. The identity of isolated bacteria was confirmed by MALDI-TOF mass spectrometry. Non-aureus staphylococci (NAS) were the most common pathogens (24.7%) and, among 36 NAS tested, 36.1% were resistant to penicillin. Thus there was high occurrence of SCM on the study farms, with relatively high penicillin resistance in NAS. Further studies are needed to identify underlying risk factors and develop an udder health control strategy for water buffalo in Bangladesh.

Resistance to ß-lactams in Streptococcus pneumoniae.

Streptococcus pneumoniae is an important causal agent of pneumonia, meningitis, sepsis, bacteremia, and otitis media. Penicillin resistance rates in S. pneumoniae have remained stable in Argentina in the last years. In the late '90s more isolates with MIC of penicillin ≥2µg/ml were observed; however, their frequency has decreased in recent years. The phenotypic expression of penicillin resistance is due to a modification in penicillin-binding proteins associated with a mosaic structure in the coding genes. The expansion of successful resistant clones varies among the different regions and is influenced by the use of antibiotics, vaccines, particularly conjugated ones, as well as population density. Parenteral treatment with high doses of penicillin G continues to be effective for the treatment of pneumonia and bacteremia, oral aminopenicillins for otitis media and sinusitis and third generation cephalosporins for meningitis.

Smart pH-Regulated Switchable Nanoprobes for Photoelectrochemical Multiplex Detection of Antibiotic Resistance Genes.

Antibiotic resistance, encoded via particular genes, has become a major global health threat and substantial burden on healthcare. Hence, the facile, low-cost, and precise detection of antibiotic resistance genes (ARGs) is crucial in the realm of human health and safety, especially multiplex sensing assays. Here, a smart pH-regulated switchable photoelectrochemical (PEC) bioassay has been created for ultrasensitive detection of two typical subtypes of penicillin resistance genes bla-CTX-M-1 (target 1, labeled as TDNA1) and bla-TEM (target 2, labeled as TDNA2), whereby pH-responsive antimony tartrate (SbT) complex-grafted silica nanospheres are ingeniously adopted as signal DNA1 tags (labeled as SDNA1-SbT@SiO2NSs). The operations of the PEC bioassay depend on the switchable dissociation of the pH-responsive SDNA1-SbT@SiO2NSs complex under the external pH stimuli, thus initiating the pH-regulated release of ions pre-embedded in sandwich-type DNA nanoassemblies. At acidic conditions, the dissociation of SDNA1 tags (ON state) triggers the release of the embedded SbO+. Under alkaline conditions, the dissociation of SDNA1 tags is inhibited (OFF state). The detection of TDNA2 was achieved via DNA hybridization-triggered metal ion release. The unwinding of the introduced hairpin T-Hg2+-T fragment, hybridized with the second anchored signal DNA (SDNA2), ignites the release of Hg2+. The released SbO+ or Hg2+ ions would trigger the formation of Sb2S3/ZnS or HgS/ZnS heterostructure through ion-exchange with the photosensitive ZnS layer, giving rise to the amplified photocurrents and eventually realizing the ultrasensitive detection of penicillin resistance genes subtypes, bla-CTX-M-1 and bla-TEM. The as-fabricated pH-regulated PEC bioassay, smartly integrating the pH-responsive intelligent unit as SDNA tags, pH-regulated release of embedded ions, and the subsequent ion-exchange-based signal amplification strategy, exhibits high sensitivity, specificity, low-cost, and ease of use for multiplex detection of ARGs. It can be successfully used for measuring bla-CTX-M-1 and bla-TEM in real E. coli plasmids, demonstrating great promise for developing a new class of genetic point-of-care devices.

Heterogeneity of penicillin-non-susceptible group B streptococci isolated from a single patient in Germany.

OBJECTIVES: Streptococcus agalactiae [group B streptococci (GBS)] have been considered uniformly susceptible to penicillin. However, increasing reports from Asia and North America are documenting penicillin-non-susceptible GBS (PRGBS) with mutations in pbp genes. Here we report, to the best of our knowledge, the first two PRGBS isolates recovered in Europe (AC-13238-1 and AC-13238-2), isolated from the same patient. METHODS: Two different colony morphologies of GBS were noted from a surgical abscess drainage sample. Both were serotyped and antimicrobial susceptibility testing was performed by different methodologies. High-throughput sequencing was done to compare the isolates at the genomic level, to identify their capsular type and ST, to evaluate mutations in the pbp genes and to compare the isolates with the genomes of other PRGBS isolates sharing the same serotype and ST. RESULTS: Isolates AC-13238-1 and AC-13238-2 presented MICs above the EUCAST and CLSI breakpoints for penicillin susceptibility. Both shared the capsular type Ia operon and ST23. Genomic analysis uncovered differences between the two isolates in seven genes, including altered pbp genes. Deduced amino acid sequences revealed critical substitutions in PBP2X in both isolates. Comparison with serotype Ia clonal complex 23 PRGBS from the USA reinforced the similarity between AC-13238-1 and AC-13238-2, and their divergence from the US strains. CONCLUSIONS: Our results support the in-host evolution of ß-lactam-resistant GBS, with two PRGBS variants being isolated from one patient.

Performance of penicillinase detection tests in Staphylococcus epidermidis: comparison of different phenotypic methods.

BACKGROUND: Staphylococcus epidermidis is the leading coagulase negative staphylococci (CoNS) species associated with healthcare associated infections. In order to de-escalate antimicrobial therapy, isolates of S. epidermidis lacking the blaZ gene should be eligible for targeted antimicrobial therapy. However, testing the susceptibility of coagulase negative staphylococci (CoNS) to penicillin G is no longer recommended by EUCAST, given the low performances for penicillinase detection in CoNS. The objective of this work was to determine a phenotypic method with high performance for detecting penicillinase production in S. epidermidis. RESULTS: Four techniques for the detection of penicillinase production (disk diffusion, zone edge test, nitrocefin test, Minimal Inhibitory Concentration (MIC) by automated system Vitek2®) were evaluated on 182 S. epidermidis isolates, using identification of blaZ gene by PCR as the reference method. The performance of the methods for penicillinase detection was compared by the sensitivity, the specificity, the negative predictive value and the positive predictive value, and with Cohen's kappa statistical test. Among the 182 S. epidermidis included in this study, 55 carried the blaZ gene. The nitrocefin test, characterized by a poor sensitivity (91%), was therefore excluded from S. epidermidis penicillinase detection. The algorithm proposed here for the penicillinase detection in S. epidermidis involved two common antimicrobial susceptibility techniques: disk diffusion method and MIC by Vitek2® system. Disk diffusion method, interpreted with a 26 mm breakpoint for penicillin G, was associated with a high sensitivity (98%) and specificity (100%). This method was completed with zone edge test for S. epidermidis with penicillin G diameter from 26 to 35 mm (sensitivity of 98%). The Vitek2® system is associated with a low sensitivity (93%) and a high specificity (99%) This low sensitivity is associated with false negative results, in isolates with 0.12 mg/L Penicillin G MIC values and blaZ positive. Thus for penicillin G MIC of 0.06 mg/L or 0.12 mg/L, a second step with disc diffusion method is suggested. CONCLUSIONS: According to our results, the strategy proposed here allows the interpretation of penicillin G susceptibility in S. epidermidis isolates, with an efficient detection of penicillin G resistance.