Altered Penicillin-binding Protein Research Articles

Resistance to aztreonam-avibactam among clinical isolates of Escherichia coli is primarily mediated by altered penicillin-binding protein 3 and impermeability

This study was conducted to investigate decreased susceptibility (minimum inhibitory concentrations [MICs] 0.25–4 mg/L) and resistance (MICs > 4 mg/L) to aztreonam-avibactam (ATM-AVI). Contemporary non-replicate clinical isolates of carbapenemase-producing Escherichia coli (CP-EC) (n=90) and ESBL-producing E. coli (EP-EC) (n=12) were used. CP-EC belonged to 25 distinct sequence types (STs) and all EP-EC belonged to ST405. All strains were isolated from 2019 to 2022 at the Karolinska University Laboratory, Stockholm, Sweden. ATM-AVI MICs were determined using broth microdilution. The EUCAST epidemiological cut-off value of 0.125 mg/L was used to define the wild type (WT). Whole-genome sequences (Illumina) were analysed for detecting resistance determinants among WT vs. non-WT isolates. Among 102 isolates, 40 (39%) and 62 (61%) were WT and non-WT, respectively. Among non-WT isolates, resistance was noted for 20 and decreased susceptibility for 42. Resistance was observed among 14/47 New Delhi metallo-β-lactamase (NDM)-producers, 5/43 OXA-48 group producers, and 1/12 EP-EC. Decreased susceptibility was observed among 29/47 NDM, 13/43 OXA-48 group, and 3/12 EP-EC. Resistant isolates predominantly belonged to ST405, followed by STs 410, 361, 167, 617, and 1284. Penicillin-binding protein 3 (PBP3) inserts (YRIK/YRIN) were observed in 20/20 and CMY-42 in 5/20 resistant isolates. Several mutations in the ftsI (encoding PBP3) and regulatory genes of outer membrane proteins (OmpC and OmpF) and efflux pumps (AcrAB-TolC) were detected. A ≥ 2-fold reduction in MICs was observed among 20/20 vs. 7/20 isolates tested in the presence of the membrane permeabiliser, polymyxin B nanopeptide (PMBN) and efflux inhibitor, phenylalanine arginine β-naphthylamide (PAβN), respectively. In conclusion, resistance to ATM-AVI is a result of interplay of various determinants, including target alterations, deactivating enzymes, and decreased permeability.

Characterization of Ceftriaxone-Resistant Haemophilus influenzae Among Korean Children.

Haemophilus influenzae is a frequently encountered pathogen responsible for respiratory tract infections in children. Following the detection of ceftriaxone-resistant H. influenzae at our institution, we aimed to investigate the resistance mechanisms of ceftriaxone in H. influenzae, with a particular focus on alterations in penicillin-binding protein 3 (PBP3) and β-lactamase production. Among H. influenzae isolates collected at Asan Medical Center Children's Hospital from March 2014 to April 2019, ceftriaxone-resistant strains by the disk-diffusion test were included. Ceftriaxone minimum inhibitory concentrations (MICs) were determined using the E-test according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The presence of β-lactamase was assessed through cefinase test and TEM-1/ROB-1 polymerase chain reaction (PCR). PBP3 alterations were explored via ftsI gene sequencing. Out of the 68 collected strains, 21 exhibited resistance to ceftriaxone in disk diffusion tests. Two strains were excluded due to failed subculture. Among 19 ceftriaxone-resistant H. influenzae isolates, eighteen were non-typeable H. influenzae, and twelve were positive for TEM-1 PCR. Isolates were classified into groups II (harboring only N526K, n = 3), III (N526K+S385T, n = 2), III+ (S385T+L389F+N526K, n = 11), and III-like+ (S385T+L389F+R517H, n = 3) according to the PBP3 alteration pattern. With a median ceftriaxone MIC of 0.190 mg/L (range, 0.008-0.750), the median ceftriaxone MIC was the highest in group III-like+ (0.250 mg/L), followed by groups III+ (0.190 mg/L), III (0.158 mg/L), and II (0.012 mg/L). All three strains belonging to group II, which did not harbor the S385T substitution, had ceftriaxone MICs of ≤ 0.125 mg/L. The emergence of ceftriaxone-resistant H. influenzae with ceftriaxone MIC values of up to 0.75 mg/L was observed even in children in South Korea, with most associated with S385T and L389F substitutions. The N526K mutation alone does not significantly impact ceftriaxone resistance. Further large-scale studies are essential to investigate changes in antibiotic resistance patterns and factors influencing antibiotic resistance in H. influenzae isolated from pediatric patients in Korea.

Cheminformatics identification of phenolics as modulators of penicillin-binding protein-3 of Pseudomonas aeruginosa towards interventive antibacterial therapy

Antibacterial resistance to β-lactams in microorganisms has been attributed majorly to alterations in penicillin-binding proteins (PBPs) coupled with β-lactams’ inactivation by β-lactamase. Consequently, the identification of a novel class of therapeutics with improved modulatory action on the PBPs is imperative and plant secondary metabolites, including phenolics, have found relevance in this regard. For the first time in this study, the over 10,000 phenolics currently known were computationally evaluated against PBP3 of Pseudomonas aeruginosa, a superbug implicated in several nosocomial infections. In doing this, a library of phenolics with an affinity for PBP3 of P. aeruginosa was screened using structure-activity relationship-based pharmacophore and molecular docking approaches. Subsequent thermodynamic screening of the top five phenolics with higher docking scores, more drug-likeness attributes, and feasible synthetic accessibility was achieved through a 120 ns molecular dynamic (MD) simulation. Four of the top five hits had higher binding free energy than cefotaxime (−18.72 kcal/mol), with catechin-3-rhamside having the highest affinity (−28.99 kcal/mol). All the hits were stable at the active site of the PBP3, with catechin-3-rhamside being the most stable (2.14 Å), and established important interactions with Ser294, implicated in the catalytic activity of PBP3. Also, PBP3 became more compact with less fluctuation of the active site amino acid residues following the binding of the hits. These observations are indicative of the potential of the test compounds as PBP3 inhibitors, with catechin-3-rhamside being the most prominent of the compounds that could be further improved for enhanced druggability against PBP3 in vitro and in vivo. Communicated by Ramaswamy H. Sarma

Relationship between Penicillin-Binding Proteins Alterations and β-Lactams Non-Susceptibility of Diseased Pig-Isolated Streptococcus suis.

Streptococcus suis is a zoonotic pathogen causing disease in both animals and humans, and the emergence of increasingly resistant bacteria to antimicrobial agents has become a significant challenge globally. The objective of this study was to investigate the genetic basis for declining susceptibility to penicillin and other β-lactams among S. suis. Antimicrobial susceptibility testing and penicillin-binding proteins (PBP1a, PBP2a, PBP2b, and PBP2x) sequence analysis were performed on 225 S. suis isolated from diseased pigs. This study found that a growing trend of isolates displayed reduced susceptibility to β-lactams including penicillin, ampicillin, amoxicillin/clavulanic acid, and cephalosporins. A total of 342 substitutions within the transpeptidase domain of four PBPs were identified, of which 18 substitutions were most statistically associated with reduced β-lactams susceptibility. Almost all the S. suis isolates which exhibited penicillin-non-susceptible phenotype (71.9%) had single nucleotide polymorphisms, leading to alterations of PBP1a (P409T) and PBP2a (T584A and H588Y). The isolates may manifest a higher level of penicillin resistance by additional mutation of M341I in the 339STMK active site motif of PBP2x. The ampicillin-non-susceptible isolates shared the mutations in PBP1a (P409T) and PBP2a (T584A and H588Y) with additional alterations of PBP2b (T625R) and PBP2x (T467S). The substitutions, including PBP1a (M587S/T), PBP2a (M433T), PBP2b (I428L), and PBP2x (Q405E/K/L), appeared to play significant roles in mediating the reduction in amoxicillin/clavulanic acid susceptibility. Among the cephalosporins, specific mutations strongly associated with the decrease in cephalosporins susceptibility were observed for ceftiofur: PBP1a (S477D/G), PBP2a (E549Q and A568S), PBP2b (T625R), and PBP2x (Q453H). It is concluded that there was genetically widespread presence of PBPs substitutions associated with reduced susceptibility to β-lactam antibiotics.

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

Haemophilus influenzae is a causative agent of serious infections, especially among children. β-lactam antibiotics are commonly used for the treatment of these infections. Among H. influenzae isolates, β-lactam resistance is due to the presence of β-lactamase, or to mutations in the ftsI gene that generate altered PBP3 (penicillin-binding protein 3) with reduced affinity for β-lactams (BLNAR-β-lactamase-negative, ampicillin-resistant). Wild-type ftsI gene encoding for PBP3 was amplified in whole from β-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 β-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 β-lactam susceptibility in H. influenzae.

Sulopenem: An Intravenous and Oral Penem for the Treatment of Urinary Tract Infections Due to Multidrug-Resistant Bacteria.

Sulopenem: An Intravenous and Oral Penem for the Treatment of Urinary Tract Infections Due to Multidrug-Resistant Bacteria.

Investigation of mechanisms responsible for decreased susceptibility of aztreonam/avibactam activity in clinical isolates of Enterobacterales collected in Europe, Asia and Latin America in 2019.

BackgroundThe combination aztreonam/avibactam is currently under Phase 3 trials for the treatment of serious infections caused by Gram-negative bacteria including those with MBLs.ObjectivesTo investigate the resistance mechanisms in Enterobacterales exhibiting aztreonam/avibactam MICs of ≥4 mg/L.MethodsAmong 8787 Enterobacterales, 17 (0.2%) isolates exhibited an aztreonam/avibactam MIC of ≥4 mg/L. Isolates were sequenced and screened for β-lactamases. Sequences of porins, penicillin-binding protein 3 (PBP3) and expression levels of AmpC and AcrA were evaluated.ResultsEleven (11/4154 isolates; 0.26%) Escherichia coli, three (3/1981; 0.15%) Klebsiella pneumoniae and three (3/628; 0.5%) Enterobacter cloacae were identified. All E. coli showed either an ‘YRIK’ or ‘YRIN’ insertion in PBP3. In general, these isolates carried blaCMY and/or blaCTX-M variants, except for one isolate from Korea that also produced NDM-5 and one isolate from Turkey that produced OXA-48. Two DHA-1-producing K. pneumoniae overexpressed acrA and had a premature stop codon in either OmpK35 or OmpK36, whereas a third K. pneumoniae carried blaPER-2 and had a premature stop codon in OmpK35. All three E. cloacae expressed AmpC at levels ≥570-fold, but sequence analysis did not reveal known amino acid alterations associated with decreased avibactam binding or increased hydrolysis of β-lactams. Minor amino acid polymorphisms within OmpC, OmpF and PBP3 were noted among the E. cloacae.ConclusionsA small number of isolates (0.2%) met the inclusion criteria. E. coli showed altered PBP3 as the most relevant resistance mechanism, whereas K. pneumoniae had multiple resistance mechanisms. Further investigations are needed to clarify resistance in E. cloacae.

Pseudomonas aeruginosa: Mechanisms of resistance to antibiotics and case analysis

Pseudomonas aeruginosa is an opportunistic pathogen, causing great concern due to the rapid increase in its resistance to antibiotics. The objective of the research was to describe the resistance mechanisms that P. aeruginosa possesses, as well as to report its behavior against antibiotics in the years 2003 to 2018 in Mexico. A retrospective and longitudinal documentary research was carried out in different digital resources referring to antibiotic resistance in P. aeruginosa. The results showed that the main resistance mechanisms of P. aeruginosa are β-lactamases, ejection pumps, mutations in porins, acquisition of plasmids, low permeability, formation of biofilms, alterations in penicillin-binding proteins, modifying enzymes of aminoglycosides and mutations in the active site. The antibiotics with the highest percentage of resistance presented between the years 2003 to 2018 were imipenen IMP, gentamicin GEN, ticarcillin / clavulanate TIC, trimethoprim / sulfamethoxazole TMS, ticarcillin TC, ceftriaxone CRO, aztreonam AZT, ceftazidime CAZ, cefepime FEP, levofloxacin LEV, chloramphenicol CL, tigecilcin TIG and ciprofloxacin CIP, with a peak of resistance between the years 2007 to 2008 and a possible downward trend in 2018. The behavior of resistance between the years 2003 to 2018 report a downward trend in the last two years.

Cranberry-Derived Proanthocyanidins Potentiate β-Lactam Antibiotics against Resistant Bacteria.

The emergence and spread of extended-spectrum β-lactamases (ESBLs), metallo-β-lactamases (MBLs), or variant low-affinity penicillin-binding proteins (PBPs) pose a major threat to our ability to treat bacterial infection using β-lactam antibiotics. Although combinations of β-lactamase inhibitors with β-lactam agents have been clinically successful, there are no MBL inhibitors in current therapeutic use. Furthermore, recent clinical use of new-generation cephalosporins targeting PBP2a, an altered PBP, has led to the emergence of resistance to these antimicrobial agents. Previous work shows that natural polyphenols such as cranberry-extracted proanthocyanidins (cPAC) can potentiate non-β-lactam antibiotics against Gram-negative bacteria. This study extends beyond previous work by investigating the in vitro effect of cPAC in overcoming ESBL-, MBL-, and PBP2a-mediated β-lactam resistance. The results show that cPAC exhibit variable potentiation of different β-lactams against β-lactam-resistant Enterobacteriaceae clinical isolates as well as ESBL- and MBL-producing E. coli We also discovered that cPAC have broad-spectrum inhibitory properties in vitro on the activity of different classes of β-lactamases, including CTX-M3 ESBL and IMP-1 MBL. Furthermore, we observe that cPAC selectively potentiate oxacillin and carbenicillin against methicillin-resistant but not methicillin-sensitive staphylococci, suggesting that cPAC also interfere with PBP2a-mediated resistance. This study motivates the need for future work to identify the most bioactive compounds in cPAC and to evaluate their antibiotic-potentiating efficacy in vivoIMPORTANCE The emergence of β-lactam-resistant Enterobacteriaceae and staphylococci compromises the effectiveness of β-lactam-based therapy. By acquisition of ESBLs, MBLs, or PBPs, it is highly likely that bacteria may become completely resistant to the most effective β-lactam agents in the near future. In this study, we described a natural extract rich in proanthocyanidins which exerts adjuvant properties by interfering with two different resistance mechanisms. By their broad-spectrum inhibitory ability, cranberry-extracted proanthocyanidins could have the potential to enhance the effectiveness of existing β-lactam agents.

MRSA/MSSA causing infections: prevalence of mecA gene

Abstract Introduction Staphylococcus aureus is part of the human flora, present in the skin and mucous membranes but can become pathogenic, causing a wide spectrum of infections that were initially treated with penicillin. However, were observed some strains with resistance to this antibiotic and, therefore was developed a new antibiotic, the methicillin. After its introduction, arose the first S. aureus with resistance to methicillin (MRSA) due to the presence of a gene known as mecA that encodes an altered penicillin binding protein (PBP2a). In Europe, it is estimated that MRSA are associated to 44% of hospital acquired infections and its mortality rate is around 20%. Objectives Prevalence of MRSA strains in different types of infection in Coimbra district. Methodology Were analysed a total of 539 isolates of S. aureus previously characterized to the antibiotic susceptibility profile in the Hospital and University Center of Coimbra. Through the minimum inhibitory concentration (MIC) of oxacillin we classified our strains into MRSA and S. aureus methicillin-sensitive (MSSA); simultaneously, the mecA gene was detected by Polymerase Chain Reaction (PCR). Results Of the 539 isolates, 49% were considered MRSA and 51% MSSA. All MRSA isolates express the mecA gene, but from the total of 276 MSSA, 191 show this gene but do not express it. MRSA isolates were mostly from respiratory tract samples (48%) and blood cultures (21%) while MSSA were isolated in skin and soft tissue samples (35%). Conclusion MRSA are considered one of the primary pathogens for the development of pneumonia and septicaemia due to its highly virulent potential and the increasing expression of genetic determinants of antimicrobial resistance. Therefore, infections caused by MRSA continue with highly representability in the clinical context and their dissemination is a public health problem.

High prevalence of antimicrobial resistance in non-vaccine serotypes of non-invasive/colonization isolates of Streptococcus pneumoniae: A cross-sectional study eight years after the licensure of conjugate vaccine in Japan

BackgroundMultidrug resistance (MDR) in non-vaccine serotypes (NVTs)-Streptococcus pneumoniae is a global public health concern after the widespread use of pneumococcal conjugate vaccines (PCVs). The present study aimed to analyze the prevalence of serotypes and antimicrobial susceptibilities of non-invasive/colonization isolates of S. pneumoniae eight years after the introduction of PCV in Japan. MethodsA total of 545 non-invasive pneumococcal isolates (460 children, 85 adults) obtained from July 2018 to January 2019 were studied. All isolates were tested for susceptibility to ten antimicrobials and were characterized for serotypes, penicillin binding protein (PBP) genotypes, and macrolide resistance genes. ResultsAmong children, 95.0% of isolates belonged to non-13-valent pneumococcal conjugate vaccine (PCV13) serotypes (NVTs), with 15A (15.0%) being dominant, followed by 35B (12.2%), 23A (11.1%), 15B (9.8%), and 15C (9.6%). In contrast, serotype 3 (18.8%) was the most prevalent in adults, while 15A, 10A, and 23A were also common. Serotypes covered by PCV13 and 23-valent pneumococcal polysaccharide vaccine (PPSV23) in all the adult isolates were 25.9% and 48.2%, respectively. High resistance rates were observed against erythromycin, tetracycline, and clindamycin (≥62.4% children, ≥58.8% adults). Penicillin-resistant and penicillin-intermediate isolates represented 2.2% and 33.8% of all isolates, respectively. Most isolates of the three dominant NVTs 15A, 35B, and 23A were non-susceptible to penicillin. Overall, multiple drug resistance (MDR) was detected in 69.0% of all isolates, including dominant NVTs 15A, 35B, 23A, 15B, and 15C. Among all the isolates, 81.8% harbored at least one altered PBP genes, and erm(B), mef(A/E) and both these genes were found in 68.2%, 23.3%, and 6.8%, respectively. High prevalence (>90%) of erm(B) was observed in serogroup 15, and serotypes 23A, 33F, and 12F. ConclusionThe present study revealed the high prevalence of NVTs 15 and 23A, and 35B showing MDR, suggesting their spread after introduction of routine PCV immunization.

Polymorphism of ftsI gene in Haemophilus influenzae and emergence of cefotaxime resistance in two Tunisian hospitals.

The decreased affinity to β-lactams in Haemophilus influenzae is usually caused by specific alterations in penicillin-binding protein 3 due to varieties of substitutions in ftsI gene. This study aimed to characterize the polymorphism of ftsI gene in 19 H. influenzae strains, isolated between 2014 and 2016 (different resistance phenotypes to β-lactams (n = 9) and susceptible strains (n = 10) used for comparative purposes).All strains were characterized for capsular type by PCR and agglutination tests and for β-lactam resistance by amplification and sequencing of ftsI. Biotyping and clonality were performed by API-NH and pulsed-field gel electrophoresis, respectively.Four strains were β-lactamase-negative ampicillin-resistant and five were β-lactamase-positive clavulanic-acid-resistant. One strain from each group was resistant to cefotaxime. Our isolates belonged mainly to biotype IV and I and were non-typeable and genetically unrelated. According to mutation profiles of their ftsI, strains were classified as group I (n = 3), group II (n = 4), group–III–like (n = 1) and group III (n = 1). All group II strains were further classified as subgroup IIb, except for one strain, which harboured a new mutation (N422I). Ampicillin MICs of β-lactamase-negative ampicillin-resistant strains were 6 to 12 times the MICs of susceptible strains. Only blaTEM-1 was detected in β-lactamase-positive clavulanic-acid-resistant strains, and was responsible for high MICs for ampicillin (>256 mg/L), whatever the ftsI mutational resistance group.The emergence of cefotaxime-resistant isolates in our country is a matter of concern and requires strict surveillance and rationalization of antibiotic use to preserve these molecules.

Methicillin resistant Staphylococcus aureus: a review of the present Indian scenario and drug treatment

Methicillin-resistant strains of S. aureus cause infections that are associated with higher mortality rates and increased length of hospital stays, than infections caused by methicillin-susceptible strains. The mechanism by which MRSA strains develop resistance is by the production of an altered penicillin-binding protein (PBP), which has a decreased affinity for most semisynthetic penicillins. MRSA has increased in prevalence and has become endemic in the Indian subcontinent, with incidence varying from 25% in Western regions to 50% in Southern India. A multi-centric study conducted across 15 tertiary care centers in India from 2008 to 2009 showed overall prevalence of MRSA infection to be 41% among S. aureus isolates, and recent Indian studies from 2014 and 2018 have also suggested an increasing prevalence of MRSA. Transmission is mainly horizontal and concerns high risk populations (patients with prolonged hospital stay, on haemodialysis, receiving cancer treatment or specific medications that affect immune function, intravenous drug users and individuals who have had surgery in the recent past. Community Acquired MRSA has emerged as significant pathogen responsible for potentially fatal infections including life-threatening pneumonia, necrotizing fasciitis, endocarditis, osteomyelitis, severe sepsis, and toxic shock syndrome. Diagnosis is mainly by gram stain, culture and quantitative PCR. Newer techniques include Multi-locus sequence typing (MLST), Pulsed-field gel electrophoresis (PFGE), Bacteriophage typing, spa locus typing and SCCmec typing. Although Vancomycin was previously considered the definitive treatment for serious MRSA infections, the emergence of less-susceptible strains, poor clinical outcomes, and increased nephrotoxicity with high-dose therapy have deterred its use as first-line therapy in many cases. Although a number of new and effective antimicrobial agents are now available for the treatment of MRSA, their exact role and choice of agent needs to be well-defined.

Tailoring Antimicrobial Susceptibility Testing to Individual Species of Coagulase-Negative Staphylococci: Next Up, Staphylococcus epidermidis.

Accurate detection of methicillin resistance among staphylococci is vital for patient care. Methicillin resistance is most commonly mediated by acquisition of the mecA gene, which encodes an altered penicillin binding protein, PBP2a. Application of phenotypic methods to detect mecA-mediated beta-lactam resistance in staphylococci is becoming more complex as species-specific differences are identified among coagulase-negative staphylococci (CoNS). Previously, interpretative criteria and antimicrobial susceptibility testing (AST) methods specific to the CoNS group were used to evaluate Staphylococcus epidermidis A manuscript by S. N. Naccache, K. Callan, C.-A. D. Burnham, M. A. Wallace, et al. (J Clin Microbiol 57:e00961-19, 2019, https://doi.org/10.1128/JCM.00961-19) details experiments revealing that S. epidermidis, the most common clinically isolated CoNS, requires tailored use of previously described methods and interpretive criteria to reliably identify the presence of mecA-mediated methicillin resistance.

The Continuing Threat of Methicillin-Resistant Staphylococcus aureus

Staphylococcus aureus has been an exceptionally successful pathogen, which is still relevant in modern age-medicine due to its adaptability and tenacity. This bacterium may be a causative agent in a plethora of infections, owing to its abundance (in the environment and in the normal flora) and the variety of virulence factors that it possesses. Methicillin-resistant S. aureus (MRSA) strains—first described in 1961—are characterized by an altered penicillin-binding protein (PBP2a/c) and resistance to all penicillins, cephalosporins, and carbapenems, which makes the β-lactam armamentarium clinically ineffective. The acquisition of additional resistance determinants further complicates their eradication; therefore, MRSA can be considered as the first representative of multidrug-resistant bacteria. Based on 230 references, the aim of this review is to recap the history, the emergence, and clinical features of various MRSA infections (hospital-, community-, and livestock-associated), and to summarize the current advances regarding MRSA screening, typing, and therapeutic options (including lipoglycopeptides, oxazolidinones, anti-MRSA cephalosporins, novel pleuromutilin-, tetracycline- and quinolone-derivatives, daptomycin, fusidic acid, in addition to drug candidates in the development phase), both for an audience of clinical microbiologists and infectious disease specialists.

Cefotaxime-non-susceptibility of Haemophilus influenzae induced by additional amino acid substitutions of G555E and Y557H in altered penicillin-binding protein 3

Cefotaxime-non-susceptible Haemophilus influenzae has rarely been isolated from clinical specimens. Although several reports have shown that amino acid (AA) alteration in penicillin-binding protein 3 (PBP3), encoded by the ftsI gene, reduces activity of cefotaxime, precise mechanisms conferring the non-susceptibility have been unclear. We analyzed the ftsI gene of two clinically isolated cefotaxime-non-susceptible H. influenzae strains, 16-11 and 20-07 (minimum inhibitory concentrations [MICs]: 16 and 8 μg/mL, respectively), and found that their deduced AA sequences of PBP3 included two AA substitutions of G555E and Y557H in addition to previously described AA alterations. To clarify whether the two additional substitutions are requisite for cefotaxime non-susceptibility, we produced transformants of Rd KW20 (cefotaxime MIC: ≤0.06 μg/mL) with the ftsI gene of 16-11. Cefotaxime MICs against transformants M1 and M2, of which deduced PBP3s were altered with that of 16-11 entirely and partially (only the N-terminal side up to the AA position 519), were 8 and 0.25 μg/mL, respectively. We also produced M2-555/7 through site-directed mutagenesis inducing additional substitutions of G555E and Y557H into the PBP3 of M2, against which cefotaxime MIC was 8 μg/mL. These findings show that the additional substitutions of G555E and Y557H in PBP3 with previously described alterations cause cefotaxime non-susceptibility. An additional substitution of either G555E or Y557H alone in altered PBP3 reduced cefotaxime activity but the elevation of MICs were within the category of susceptibility. To our knowledge, this is the first study clarifying a genetic factor in the PBP3 causing cefotaxime non-susceptibility among H. influenzae strains.

Association Between Pneumococcal Surface Protein A Family and Genetic/Antimicrobial Resistance Traits of Non-Invasive Pneumococcal Isolates from Adults in Northern Japan.

Pneumococcal isolates from adult patients in northern Japan in 2016 were subjected to molecular investigation related to pneumococcal surface protein A (PspA) and drug resistance determinants. Of the 51 isolates, serotype 3/ST180 was the most prevalent (17.6%), followed by 35B (ST2755/ST558) (11.8%) and 15A (ST63/ST7874/ST13068/ST13785) (9.8%). Coverage of serotypes by 13-valent conjugate vaccine and 23-valent polysaccharide vaccine was 27.5% and 49%, respectively. All the isolates expressed PspA family 1 or 2 (51% and 49%, respectively). Each serotype was associated with either of the PspA families (e.g., serotype 3, PspA family 1; serotypes 35B and 15A, PspA family 2). Multidrug resistance (MDR) was found in 84.3% of the isolates. Minimum of one altered penicillin-binding protein gene was detected in 82.4% of isolates, indicating 25.5% non-susceptibility to penicillin. Serotypes 15A and 35B were predominant and demonstrated MDR. An isolate of serotype 15A/ST13785 (single-locus variant of ST242) was resistant to fluoroquinolones associated with double mutation in the quinolone resistance-determining regions of gyrA and parC. The present study indicates the spread of MDR pneumococci represented by isolates of serotypes 3, 15A, and 35B, and prevalence of both PspA family 1 and 2 in isolates obtained from adult patients.

Relationship Between Different Resistance Mechanisms and Virulence in Acinetobacter baumannii.

Aim: This study analyzed the virulence of several Acinetobacter baumannii strains expressing different resistance mechanisms using the Caenorhabditis elegans infection model. Results: Strains susceptible/resistant to carbapenems (presenting class D (OXA-23, OXA-24), class B metallo-β-lactamase (MBL) (NDM-1), penicillin binding protein (PBP) altered and decreased expression of Omp 33-36 kDa) and isogenic A. baumannii strains susceptible/resistant to colistin (presenting loss of lipopolysaccharide (LPS) and pmrA mutations) were included to evaluate the virulence using the C. elegans infection model. The nematode killing assay, bacterial ingestion in worms, and bacterial lawn avoidance assay were performed with the Fer-15 mutant line. A. baumannii strains generally presented low virulence, showing no difference between carbapenem-resistant strains (expressing class D, MBLs, or altered PBP) and their isogenic susceptible strains. In contrast, the absence of the Omp 33-36 kDa protein in the knockout was associated with a decrease of virulence, and a significant difference was observed between colistin-resistant mutants and their susceptible counterpart when the mechanism of resistance was associated with the loss of LPS but not with its modification. Conclusions: Resistance to carbapenems in A. baumannii associated with the production of OXA-type or NDM-type enzymes does not seem to affect their virulence in the C. elegans infection model. In contrast, the presence of Omp 33-36 kDa, and high level resistance to colistin related with the loss of LPS, might contribute with the virulence profile in A. baumannii.

The detection of altered penicillin-binding protein 2b, autolysin, and pneumolysin genes in Streptococcus pneumoniae colonizing the upper respiratory tract of healthy schoolchildren in Puducherry

Background: Infections due to Streptococcus pneumoniae are responsible for morbidity and mortality in a large proportion of children in developing countries where childhood vaccination has not been widely implemented. S. pneumoniae colonizing the upper respiratory tract of children as a normal commensal is a potential pathogen when it crosses the mucosal barrier and enters the bloodstream. Aims and Objectives: The aim of the study was to analyze the carriage rate of pneumococci among healthy school children and in doing so to analyze the presence of penicillin-binding protein 2b (pbp2b) gene which is responsible for penicillin resistance using polymerase chain reaction. Methods: Nasopharyngeal swabs were collected from school children and processed according to standard microbiological procedures. The genes for penicillin resistance were detected using PCR. Results: The carriage rate was 32.2% and about 14 strains had the pbp2b gene. Conclusion: Pneumococcal carriage among children is important as they can cross the mucosal barrier and cause infection. Constant follow up of the carriage rate have to be performed to prevent further spread.

2004. Clinical Application of Xpert SA Nasal Complete for Direct Detection of Staphylococcus aureus and Methicillin-Resistant Staphylococcus aureus in Nasal Swabs in Pediatric Care Setting

Background Staphylococcus aureus (SA) is a major human pathogen, causing a variety of nosocomial and community-acquired infections. Nasal carriers of SA are at increased risk for healthcare associated infections with this organism. Timely detection of SA and Methicillin-resistant Staphylococcus aureus (MRSA) and decolonization of pre-surgical patients carrying SA are of importance in infection prevention. We sought to evaluate the clinical performance of the Xpert SA Nasal Complete assay (Xpert SA) for detection of SA and MRSA in the nasal specimens from pediatric patients.MethodsA total of 504 nasal specimens were collected in the Copan dual swab systems from patients with ages between 0 to 61 years with 91.9% patients ≤21 year-old (n = 463). For each sample, one swab was tested with Xpert SA. The second swab was plated onto Blood agar, Mannitol salt agar and ChromID™ MRSA plate and incubated at 35°C in non-CO2 incubator. The identification of SA and MRSA was compared between Xpert SA and the culture results. Methicillin resistance was determined using conventional methods (susceptibility testing or detection of altered penicillin binding protein).ResultsWhen compared with culture for the identification of SA (n = 481), there was an agreement of 95.0% with sensitivity and specificity being 95.6% and 94.8%, respectively. Among those culture-confirmed and Xpert SA positive samples (n = 131), concordance between Xpert SA and conventional methods for detection of methicillin resistance was 97.0% with sensitivity and specificity being 100% and 96.3%, respectively. Four culture-confirmed methicillin-susceptible SA (MSSA) were identified as MRSA by Xpert SA. Among 504 nasal specimens, 23 (4.6%) samples had invalid or instrument failure results. Nasal swabs collected from pediatric patients (≤21-year-old) had a higher invalid/instrument failure rate (5.0%) than those from adults (0%) (P < 0.001).ConclusionXpert SA Nasal Complete assay provides a rapid and sensitive method to detect and differentiate between MSSA and MRSA colonization. The higher invalid rate in pediatric patients and misidentification of MSSA as MRSA by Xpert SA warrant the confirmation by bacterial culture and conventional susceptibility test.Disclosures A. Leber, Nationwide Children’s Hospital: Research Contractor, Research support.