Β-lactam Resistance Research Articles

424. Risk Factors for Infection or Colonization with Piperacillin-tazobactam (TZP) Resistant P. aeruginosa (PSA) with Increasing Degrees of β-lactam Resistance

Abstract Background Resistance to β-lactams amongst PSA is common with roughly 20% of isolates demonstrating resistance to TZP. This can lead to delays in the time to appropriate empiric therapy. While rapid diagnostics can identify PSA at earlier time points, they fail to detect common β-lactam resistance mechanisms in this pathogen. Though risk factors for TZP resistance (TZP-R) amongst PSA have been identified, co-resistance amongst traditional anti-PSA β-lactams is common, so decisions to turn to other traditional anti-PSA or novel agents in the presence of such predictors are unclear. This study aims to find predictors unique to varying degrees of β-lactam resistance amongst TZP-R PSA to inform these decisions. Methods This was a retrospective case-case-case-control study of all non-cystic fibrosis patients with PSA isolated from a culture from 2015-2021 at Michigan Medicine. Four study groups were created based on the susceptibility profile of PSA: TZP susceptible (TZP-S), resistance to TZP only (TZP-R ONLY), resistance to TZP in addition to 1 or 2 other traditional anti-PSA β-lactams (TZP-R PLUS), and resistance to all traditional anti-PSA β-lactams (pan β-lactam resistance, PBR). Bivariate and multivariate analyses were performed to compare each degree of TZP resistance to the TZP-S group. Results were then compared across models to assess uniqueness to a given resistant phenotype. Results 2365 patients were included, and results of the multivariate analyses are listed in the table. The sole predictor of TZP-R ONLY was recent ceftriaxone or cefotaxime use. Independent predictors unique to TZP-R PLUS were community acquired infection, recent ICU residence, and recent exposure to TZP or aztreonam. Most risk factors identified were either unique to PBR or shared between PBR and TZP-R PLUS including history of PSA, history of TZP-R PSA, and recent exposure to multiple antibiotics. Multivariate Models for TZP-R ONLY, TZP-R PLUS, and PBR Conclusion This analysis demonstrates that previously identified risk factors for TZP-R P. aeruginosa are predictors of PSA with greater degrees of resistance. Given these findings, consideration needs to be given to escalation to novel agents in the presence of risk factors at the time of PSA isolation, rather than to other traditional anti-PSA β-lactams. Disclosures Owen Albin, MD, Charles River Laboratory: Advisor/Consultant|Cipla Pharmaceuticals: Advisor/Consultant|Shionogi Inc: Advisor/Consultant Keith S. Kaye, MD, GSK: Advisor/Consultant|merck: Advisor/Consultant|merck: Stocks/Bonds|Qpex: Advisor/Consultant|Shionogi: Advisor/Consultant|Spero: Advisor/Consultant jason M. Pogue, PharmD, consultant to Venatorx, Merck, Shionogi, QPex Biopharma, Utility, GSK, and Entasis.: Advisor/Consultant.

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.

125. Induction of Resistance Against Antipseudomonal Agents: Comparison of Novel b-Lactam/b-Lactamase Inhibitor (BL/BLI) Combinations and Other b-lactam Agents

Abstract Background The acquisition of mutations is the main driver of β-lactam resistance in Pseudomonas aeruginosa isolates. New BL/BLIs, such as ceftazidime-avibactam (CAZ-AVI), ceftolozane-tazobactam (C/T), and imipenem-relebactam (IMI-REL), are active against most P. aeruginosa isolates from US hospitals, but the ability of these agents to induce resistance have not been explored. We subjected 8 P. aeruginosa isolates, including ATCC 27853 and 7 clinical isolates, to a 10-day serial passage with 6 antipseudomonal agents to evaluate resistance levels and mechanisms in terminal mutant strains. Fold change in MIC results from parent isolate to terminal mutant Methods Serial passaging was performed in broth microdilution (BMD) for CAZ-AVI, IMI-REL, C/T, meropenem (MEM), cefepime (FEP), and piperacillin-tazobactam (P/T). The MIC of the terminal mutants was determined after 2X passaging on drug-free agar. Parent strains and terminal mutants were subjected to short-read whole genome sequencing (WGS) at 100X coverage. Parent isolates were sequenced using long-read WGS and the data was combined with short-reading sequencing for single nucleotide polymorphism (SNP) analysis. Results Overall, IMI-REL (1- to 4-fold) and CAZ-AVI (2- to 8-fold) displayed lower fold increases in MIC values when compared to other agents tested (Figure). Of the CAZ-AVI terminal mutants, 3 displayed a nalD regulator alteration, and 1 of these had a clpA chaperone missense substitution. FEP terminal mutants exhibited alterations in ampD, mexB, and the TetR family transcriptional regulator AmrR. C/T mutants had ampG and ftsI missense alterations. MEM mutants had nalC, ftsI, and phoP missense alterations. Mutations in merR, nalC, and ampD were observed in the P/T terminal mutants. Among 2 IMI-REL terminal mutants displaying a SNP alteration, 1 displayed a nonsense mutation in pilF, a pilus forming protein. Many terminal mutants displayed alterations in genes not commonly associated to β-lactam resistance. Conclusion MEM, FEP, and P/T terminal mutants displayed high MIC values compared to those obtained after exposure to C/T, CAZ-AVI, and IMI-REL. This data might indicate a benefit of using these newer agents to prevent the emergence of high-level resistance. Disclosures Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|GSK: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support Jill Lindley, BS, AbbVie: Grant/Research Support Timothy Doyle, MS, AbbVie: Grant/Research Support John H. Kimbrough, PhD, AbbVie: Grant/Research Support|GSK: Grant/Research Support Jessica Ewald, PhD, AbbVie: Grant/Research Support Helio S. Sader, MD, PhD, AbbVie: Grant/Research Support|Cidara: Grant/Research Support|Melinta: Grant/Research Support|Nabriva Therapeutics: Grant/Research Support|Pfizer: Grant/Research Support.

325. Molecular Characterization of Antimicrobial Resistance Genes in Neisseria gonorrhoeae in Southeast Michigan

Abstract Background Antimicrobial resistance (AMR) in Neisseria gonorrhoeae (NG) is a public health crisis. Diagnosis of infection is primarily established using nucleic acid amplification technologies (NAATs) without susceptibility testing; thus rapid identification of resistance determinants is crucial. Whole-genome sequencing (WGS) is a promising alternative to current susceptibility methods. The purpose of this study was to describe AMR genes in a subset of isolates using WGS from Southeast Michigan. Methods Isolates, demographic data, and minimum inhibitory concentrations (MIC) via E-test (ceftriaxone/CRO) and broth microdilution (azithromycin/AZM) were obtained from the Michigan Department of Health and Human Services. Libraries from isolates were generated using QIAseq FX DNA library kit before sequencing on NovaSeq 6000. Reads were trimmed [Trimmomatic] and aligned to the NG genome [Burroughs-Wheeler Aligner] before processing with Samtools suite for variant detection and consensus WGS determination. AMRFinderPlus was used to categorize AMR genes and resistance-associated point mutations. Results Total of 38 isolates were analyzed. The majority were from males (63%) and Blacks (44.7%) living in Detroit City proper (47.3%) with median age 25 (range 13-56), unknown HIV status (55.2%) and sexual orientation (47.3%) (Table 1). Urine (47.3%) was the most common specimen source, followed by cervix/vagina (28.9%). More than a third had prior NG and other sexually transmitted infections. Eight resistance genes were found among the 38 isolates (Figure 1). The penA gene associated with cephalosporin resistance was found in all isolates; however, all isolates were susceptible to CRO (CLSI susceptible breakpoint MIC < 0.25). The mtrR gene associated with macrolide resistance was noted in 86.8% isolates. Among 33 isolates where AZM susceptibility was available, resistance was noted in 9 (27%) (CLSI resistance breakpoint MIC > 1). Conclusion We describe a potential utility for WGS to identify AMR genes in NG. Although numerous β-lactam resistance genes were detected in all isolates, all remained susceptible to CRO. Further research is needed to determine the extent to which genetic diversity affects phenotypic susceptibilities in NG. Disclosures All Authors: No reported disclosures.

1653. Transcriptional Variation of Adhesion and Biofilm Genes in CTX-M-15-Expressing Klebsiella pneumoniae

Abstract Background Klebsiella pneumoniae can produce a number of β-lactamases, including the cefotaximase, CTX-M-15. While classically associated with ST131 Escherichia coli, CTX-M-15 is associated with increased virulence in K. pneumoniae. Clinical isolates of K. pneumoniae have been shown to produce high levels of CTX-M-15 RNA. But, the overall transcriptomic changes due to CTX-M-15 expression are unknown. The purpose of this study was to determine if acquisition of CTX-M-15 altered the global transcriptome of K. pneumoniae in addition to decreasing β-lactam susceptibility. Methods The structural gene and native promoter of CTX-M-15 were cloned into a low copy number vector and transformed into Kp 23, a clinical isolate from the 1970s with no known acquired β-lactam resistance genes. CTX-M-15 production was confirmed by qRT PCR and western blot. MICs to cefotaxime, ceftazidime, cefepime, ceftolozane/tazobactam, ceftazidime/avibactam, meropenem, meropenem-vaborbactam, and imipenem-relebactam were determined by E-test according to CLSI guidelines. RNA-Seq analysis and bioinformatics were performed by MiGS. Results CTX-M-15 carriage led to non-susceptible MICs to cefotaxime, ceftazidime, and cefepime. CTX-M-15 production was associated with statistically significant upregulation of 204 genes and downregulation of 231 genes. Among these, the biofilm genes mrkA and two copies of mrkB were downregulated by 7.5, 4 and 3.5-fold, respectively. Additionally, ecpA, ecpB, ecpC, and ecpR – components of the E. coli Common Pilus (ECP) – were upregulated by 3, 2, 2, and 3-fold, respectively. Conclusion High-level expression of CTX-15 β-lactamase has a significant impact on the transcriptome of K. pneumoniae. These changes include a down regulation of genes associated with biofilm formation with a concomitant increase in the expression of genes associated with adherence. These data indicate that overexpression of CTX-M-15 and perhaps other β-lactamases may help promote the pathogenicity of K. pneumoniae or influence its ability to colonize the tissue it infects. This may be important during urinary tract infections when an increase in adhesion by CTX-M-15 producing isolates may be required over biofilm formation to establish an active infection. Disclosures Nancy D. Hanson, PhD, Merck: Grant/Research Support.

Enfeksiyon Etkeni Nonfermenter Gram Negatif İzolatlar ve Antibiyotik Dirençleri: Üç Yıllık Veri

Objective: This study aimed to investigate clinical non-fermenting gram-negative isolates and antibiotic resistance profiles for three years in a tertiary hospital.Materials and Methods: A total of 3817 non-fermenting gram-negative strains isolated from various cultures between January 2017 and December 2019 in Balıkesir Atatürk City Hospital were investigated retrospectively. Identification and antibiotic susceptibilities were performed using conventional methods and PhoenixTM 100 system (Becton Dickinson, MA, USA).Results: A total of 2201 (57.7%) P. aeruginosa, 1283 (33.6%) A. baumannii-calcoaceticus complex, 202 (5.3%) S. maltophilia and 131 (3.4%) B. cepacia complex strains were identified. The majority of strains were isolated from intensive care units (54.5%), followed by internal medicine (33.4%) and surgical services (12.1%). All A. baumannii-calcoaceticus complex species showed over 70% resistance to most antibiotics. In addition to β-lactam antibiotic resistance (generally over 30%), resistance to fluoroquinolones (30.4%) seemed to have particular importance. Co-trimoxazole showed below 10% resistance in S. maltophilia isolates. In B. cepacia complex, ceftazidime resistance increased in years (2018, 22.2%; 2019, 67.0%). Conclusion: The issue of antibiotic resistance cannot be won by just developing novel antimicrobials, but also by increasing the efficiency of current ones. The first step is to “diagnose” the current condition, like local surveillance studies.

In vitro and computational studies of the β-lactamase inhibition and β-lactam potentiating properties of plant secondary metabolites

β-lactam resistance in bacteria is primarily mediated through the production of β-lactamases. Among the several strategies explored to mitigate the issue of β-lactam resistance, the use of plant secondary metabolites in combination with existing β-lactams seem promising. The present study aims to identify possible β-lactam potentiating plant secondary metabolites following in vitro and in silico approaches. Among 180 extracts from selected 30 medicinal plants, acetone extract of Ficus religiosa (FRAE) bark recorded the least IC50 value of 3.9 mg/ml. Under in vitro conditions, FRAE potentiated the activity of ampicillin, which was evidenced by the significant reduction in IC50 values of ampicillin against multidrug resistant bacteria. Metabolic profiling following HR-LCMS analysis revealed the presence of diverse metabolites viz. flavonoids, alkaloids, terpenoids, etc. in FRAE. Further, ensemble docking of the FRAE metabolites against four Class A β-lactamase (SHV1, TEM1, KPC2 and CTX-M-27) showed quercetin, taxifolin, myricetin, luteolin, and miquelianin as potential inhibitors with the least average binding energy. In molecular dynamic simulation studies, myricetin formed the most stable complex with SHV1 and KPC-2 while miquelianin with TEM1 and CTX-M-27. Further, all five metabolites interacted with amino acid residue Glu166 in Ω loop of β-lactamase, interfering with the deacylation step, thereby disrupting the enzyme activity. The pharmacokinetics and ADMET profile indicate their drug-likeness and non-toxic nature, making them ideal β-lactam potentiators. This study highlights the ability of metabolites present in FRAE to act as β-lactamase inhibitors. Communicated by Ramaswamy H. Sarma

In Vitro Selection of Enterobacter cloacae with Cefepime, Meropenem, and Ceftazidime-Avibactam Generates Diverse Resistance Mechanisms

Five Enterobacter cloacae isolates were subjected to 10-day serial passage in broth microdilution with cefepime, meropenem, or ceftazidime-avibactam to evaluate increases in minimum inhibitory concentration (MIC) and resistance mechanisms after exposure. Post-exposure isolates displaying >2-fold changes from the parent isolate were analysed alongside the parent isolate. Increases in MIC were 4- to 256-fold (median: 16-fold) after cefepime exposure, 16- to 128-fold (64-fold) after meropenem, and 2- to 32-fold (8-fold) after ceftazidime-avibactam. Post-exposure isolates had diverse mechanisms, identified using a combination of short and long whole-genome sequencing. All agents selected for AmpC alterations in one isolate set. OmpC and TetA/AcrR regulator alterations were noted in meropenem and ceftazidime-avibactam post-exposure isolates of the same set. Other mutations in AmpC were noted when isolates were exposed to cefepime or ceftazidime-avibactam. A premature stop codon in the cell division inhibitor protein, MioC was observed when one parent isolate was exposed to any of the agents, indicating a cell persistence mechanism. Mutations in less common transporter systems and protein synthesis components were also noted. All agents showed cross-resistance to other β-lactams and resistance mechanisms were diverse, with some not usually associated with β-lactam resistance in Enterobacterales. This initial evaluation indicates that cefepime and meropenem select for isolates with higher MIC values compared to ceftazidime-avibactam. Further studies evaluating these findings should be performed for other species for which the primary β-lactam resistance mechanism is not gene acquisition. These studies should evaluate these observations in vivo to assess their translation into patient treatment policies.

Interspecies recombination, not de novo mutation, maintains virulence after β-lactam resistance acquisition in Streptococcus pneumoniae

As opposed to de novo mutation, β-lactam resistance in S.pneumoniae is often conferred via homologous recombination during horizontal gene transfer. We hypothesize that β-lactam resistance in pathogenic streptococci is restricted to naturally competent species via intra-/interspecies recombination due to invivo fitness trade-offs of de novo penicillin-binding protein (PBP) mutations. We show that de novo mutant populations have abrogated invasive disease capacity and are difficult to evolve invivo. Conversely, serially transformed recombinant strains efficiently integrate resistant oral streptococcal DNA, gain penicillin resistance and tolerance, and retain virulence in mice. Large-scale changes in pbp2X, pbp2B, and non-PBP-related genes occur in recombinant isolates. Our results indicate that horizontal transfer of β-lactam resistance engenders initially favorable or minimal cost changes invivo compared with de novo mutation(s), underscoring the importance of recombination in the emergence of β-lactam resistance and suggesting why some pathogenic streptococci lacking innate competence remain universally susceptible.

Exploitation of the antibacterial, antibiofilm and antioxidant activities of Salvadora Persica (Miswak) extract

The Salvadora persica (S. persica) L. chewing stick, usually known as miswak, is still being employed as an oral hygiene agent for plaque and gingivitis prevention. This study aims to assess the antibacterial, antibiofilm, antioxidant, and phytochemical profile of S. persica extract. The S. persica was purchased from a local market, grinded and extracted with petroleum ether. The disk diffusion, microdilution, and micro-plate assays were performed to evaluate the antibacterial and antibiofilm activities of the prepared extract at different concentrations against β-lactam resistance Streptococcus species. Free radical scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) and stable radical cationic chromophore, 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) methods were used to determine their antioxidant activity. Chromatographic and spectrometric analyses were performed using gas chromatography-mass (GC-MS) spectrometry. The minimum inhibitory concentration (MIC) of S. persica extract against β-lactam resistance Streptococcus species ranged from 6.25 to 12.5 mg/mL. The maximum suppression of biofilm formation by S. persica extract was observed at MIC with a percentage of 68.66%, against Streptococcus oralis. The S. persica extract exhibited antioxidant activity with IC50 of 20 µg/mL and 35 µg/mL from DPPH and ABTS, respectively. The phytochemical characterization showed the presence of 22 compounds with major compounds; benzyl isothiocyanate (36.21%) and n-hexadecanoic acid (27.62%). The S. persica extract exhibited antibacterial activity against β-lactam resistant Streptococcus species, showing a promising natural alternative that could be a treatment option.

Insights into the effects of pulsed antimicrobials on the chicken resistome and microbiota from fecal metagenomes

Antimicrobial resistance has become a global problem that poses great threats to human health. Antimicrobials are widely used in broiler chicken production and consequently affect their gut microbiota and resistome. To better understand how continuous antimicrobial use in farm animals alters their microbial ecology, we used a metagenomic approach to investigate the effects of pulsed antimicrobial administration on the bacterial community, antibiotic resistance genes (ARGs) and ARG bacterial hosts in the feces of broiler chickens. Chickens received three 5-day courses of individual or combined antimicrobials, including amoxicillin, chlortetracycline and florfenicol. The florfenicol administration significantly increased the abundance of mcr-1 gene accompanied by floR gene, while amoxicillin significantly increased the abundance of genes encoding the AcrAB-tolC multidrug efflux pump (marA, soxS, sdiA, rob, evgS and phoP). These three antimicrobials all led to an increase in Proteobacteria. The increase in ARG host, Escherichia, was mainly attributed to the β-lactam, chloramphenicol and tetracycline resistance genes harbored by Escherichia under the pulsed antimicrobial treatments. These results indicated that pulsed antimicrobial administration with amoxicillin, chlortetracycline, florfenicol or their combinations significantly increased the abundance of Proteobacteria and enhanced the abundance of particular ARGs. The ARG types were occupied by the multidrug resistance genes and had significant correlations with the total ARGs in the antimicrobial-treated groups. The results of this study provide comprehensive insight into pulsed antimicrobial-mediated alteration of chicken fecal microbiota and resistome.

Exploring the Antibiotic Resistance Profile of Clinical Klebsiella pneumoniae Isolates in Portugal.

While antibiotic resistance is rising to dangerously high levels, resistance mechanisms are spreading globally among diverse bacterial species. The emergence of antibiotic-resistant Klebsiella pneumoniae, mainly due to the production of antibiotic-inactivating enzymes, is currently responsible for most treatment failures, threatening the effectiveness of classes of antibiotics used for decades. This study assessed the presence of genetic determinants of β-lactam resistance in 102 multi-drug resistant (MDR) K. pneumoniae isolates from patients admitted to two central hospitals in northern Portugal from 2010 to 2020. Antimicrobial susceptibility testing revealed a high rate (>90%) of resistance to most β-lactam antibiotics, except for carbapenems and cephamycins, which showed antimicrobial susceptibility rates in the range of 23.5-34.3% and 40.2-68.6%, respectively. A diverse pool of β-lactam resistance genetic determinants, including carbapenemases- (i.e., blaKPC-like and blaOXA-48-like), extended-spectrum β-lactamases (ESBL; i.e., blaTEM-like, blaCTX-M-like and blaSHV-like), and AmpC β-lactamases-coding genes (i.e., blaCMY-2-like and blaDHA-like) were found in most K. pneumoniae isolates. blaKPC-like (72.5%) and ESBL genes (37.3-74.5%) were the most detected, with approximately 80% of K. pneumoniae isolates presenting two or more resistance genes. As the optimal treatment of β-lactamase-producing K. pneumoniae infections remains problematic, the high co-occurrence of multiple β-lactam resistance genes must be seen as a serious warning of the problem of antimicrobial resistance.

Antibiotics resistance evolution of isolated Vibrio parahaemolyticus from mariculture under the continuous culture of sub-inhibitory concentrations of Ulva fasciata hydroponic solution

The outbreak of vibriosis from Vibrio (V.) parahaemolyticus is widespread in the mariculture, and live macroalgae has been considered to be effective and eco-friendly approach for the control of vibriosis. Three V. parahaemolyticus strains with β-lactam antibiotics resistance (resistant to ampicillin (AM), amoxicillin (AMX)) were isolated from mariculture in study, and the antibiotics resistance evolution mechanism was examined at the sub-inhibitory concentration (SIC) of hydroponic solution of Ulva (U.) fasciata (HSUF). The HSUF with the highest density (20 g fresh weight U. fasciata L−1) demonstrated the strongest inhibitory rates (47.0 %–65.8 %) on the three strains during the stable phase (8–24 h) of growth curve, which indicated that the HSUF (≤20 g L−1) could be considered to be at SIC for V. parahaemolyticus strains. After continuous subculture of V. parahaemolyticus with three dilutes (1/2 (HT), 1/20 (MT) and 1/50 (LT)) of HSUF (20 g L−1), all the strains of 20th generation were still resistant to AM and AMX. However, the LT condition reduced MIC of AM (2–16 times) and AMX (0–2 times) to strains, while MT and HT showed significantly various effect of β-lactam antibiotics resistance on different strains. The biofilm formation and ROS content of V. parahaemolyticus were almost positively correlated to the concentrations of HSUF. Transcriptome sequencing analysis of a representative strain showed that the lower concentrations of HSUF caused more down-regulated DEGs of the strains, and more down-regulated (vmeA, vmeB, sapA, mrdA) DEGs of strains were related to the pathway of β-lactam antibiotics resistance at LT condition. Thus, low concentration of HSUF was seemed to have better improvement for V. parahaemolyticus strains resistant to β-lactam antibiotics, which were mainly related to the impairment of biofilm formation, ROS and efflux pump.

Genome sequencing analysis of a novel thermophilic strain Geobacillus sp. CX412.

The thermophilic spore-forming strain Geobacillus sp. CX412 was isolated from hot spring soil in Tengchong City, Yunnan Province, China. We sequenced the complete genome of Geobacillus sp. CX412 using PacBio SMRT Sequencing. Genome-scale phylogenetic analysis and average nucleotide identity (ANI) results indicated that Geobacillus sp. CX412 is a novel species in the genus Geobacillus. The metabolic potential of Geobacillus sp. CX412 based on COG, KEGG, and CAZymes analysis demonstrated that Geobacillus sp. CX412 was a highly adaptable strain with an unusually high number of 73 annotated transposons in the genome, which is relatively rare in Geobacillus. Compared with the near-derived strains, it was found that Geobacillus sp. CX412 has the unique β-lactam resistance and more active metabolism (more than 50.5-100.1%). Additionally, its genome encodes glycoside hydrolases and other genes related to lignocellulose breakdown, suggesting that Geobacillus sp. CX412 has a considerable biomass degradation potential. Thus, Geobacillus sp. CX412 is a new thermophilic bacterial species that add to the increasing repertoire of known lignocellulose degraders.

Bacterial Pathogens in the Food Industry: Antibiotic Resistance and Virulence Factors of Salmonella enterica Strains Isolated from Food Chain Links.

Salmonella is one of the most important foodborne pathogens. Fifty-three strains of Salmonella deposited in the Culture Collection of Industrial Microorganisms-Microbiological Resources Center (IAFB) were identified using molecular and proteomic analyses. Moreover, the genetic similarity of the tested strains was determined using the PFGE method. Main virulence genes were identified, and phenotypical antibiotic susceptibility profiles and prevalence of resistance genes were analyzed. Subsequently, the occurrence of the main mechanisms of β-lactam resistance was determined. Virulence genes, invA, fimA, and stn were identified in all tested strains. Phenotypic tests, including 28 antibiotics, showed that 50.9% of the strains were MDR. The tet genes associated with tetracyclines resistance were the most frequently identified genes. Concerning the genes associated with ESBL-producing Salmonella, no resistance to the TEM and CTX-M type was identified, and only two strains (KKP 1597 and KKP 1610) showed resistance to SHV. No strains exhibited AmpC-type resistance but for six Salmonella strains, the efflux-related resistance of PSE-1 was presented. The high number of resistant strains in combination with multiple ARGs in Salmonella indicates the possible overuse of antibiotics. Our results showed that it is necessary to monitor antimicrobial resistance profiles in all food chain links constantly and to implement a policy of proper antibiotic stewardship to contain or at least significantly limit the further acquisition of antibiotic resistance among Salmonella strains.

A Nonclassical Mechanism of β-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus and Its Effect on Virulence.

Methicillin-resistant Staphylococcus aureus (MRSA) is a group of pathogenic bacteria that are infamously resistant to β-lactam antibiotics, a property attributed to the mecA gene. Recent studies have reported that mutations associated with the promoter region of pbp4 demonstrated high levels of β-lactam resistance, suggesting the role of PBP4 as an important non-mecA mediator of β-lactam resistance. The pbp4-promoter-associated mutations have been detected in strains with or without mecA. Our previous studies that were carried out in strains devoid of mecA described that pbp4-promoter-associated mutations lead to PBP4 overexpression and β-lactam resistance. In this study, by introducing various pbp4-promoter-associated mutations in the genome of a MRSA strain, we demonstrate that PBP4 overexpression can supplement mecA-associated resistance in S. aureus and can lead to increased β-lactam resistance. The promoter and regulatory region of pbp4 is shared with a divergently transcribed gene, abcA, which encodes a multidrug exporter. We demonstrate that the promoter mutations caused an upregulation of pbp4 and downregulation of abcA, confirming that the resistant phenotype is associated with PBP4 overexpression. PBP4 has also been associated with staphylococcal pathogenesis, however, its exact role remains unclear. Using a Caenorhabditis elegans model, we demonstrate that strains having increased PBP4 expression are less virulent than wild-type strains, suggesting that β-lactam resistance mediated via PBP4 likely comes at the cost of virulence. IMPORTANCE Our study demonstrates the ability of PBP4 to be an important mediator of β-lactam resistance in not only methicillin-susceptible Staphylococcus aureus (MSSA) background strains as previously demonstrated but also in MRSA strains. When present together, PBP2a and PBP4 overexpression can produce increased levels of β-lactam resistance, causing complications in treatment. Thus, this study suggests the importance of monitoring PBP4-associated resistance in clinical settings, as well as understanding the mechanistic basis of associated resistance, so that treatments targeting PBP4 may be developed. This study also demonstrates that S. aureus strains with increased PBP4 expression are less pathogenic, providing important hints about the role of PBP4 in S. aureus resistance and pathogenesis.

Detection of Extended-Spectrum β-Lactamases (ESBLs) and AmpC in Class A and Class B Carbapenemase-Producing Enterobacterales.

In carbapenemase-producing Enterobacterales (CPE) additional β-lactam resistance mechanisms such as extended-spectrum-β-lactamases (ESBL) and/or AmpC-β-lactamases are generally difficult to detect by phenotypical methods. Recently, a modified version of the CLSI ESBL confirmatory combination disc diffusion (CDD) test, which involves the addition of boronic acid and EDTA on discs containing ESBL and AmpC substrates ± inhibitors, has been proposed for the detection of ESBL in class A and class B CPE. Here, the performance of the modified CDD test was evaluated using 121 genotypically characterized class A and class B CPE. Also, the effectiveness of the NG-Test CTX-M-MULTI lateral flow immunoassay was evaluated for ESBL detection. For class A CPE (n = 47), the modified CDD method exhibited an equal specificity (95.7%) and a higher sensitivity (100%) compared to the standard method (91.7%). The CTX-M-MULTI test detected ESBL in all CTX-M-type ESBL producers (n = 23), whereas it was negative for all CTX-M-type ESBL-negative isolates (n = 24). For class B CPE (n = 71), the modified method significantly improved both sensitivity (95%) and specificity (100%) in detecting ESBL compared to the standard method (17.5% sensitivity and 83.9% specificity). In comparison, the CTX-M-MULTI led to identification of ESBL in all CTX-M-ESBL-producers (n = 39) and no false-positive signal was generated with the CTX-M-type-ESBL-negative isolates (n = 30). Furthermore, the modified CDD improved the robustness of the method for AmpC detection (inconclusive results were produced in 53/57 and 10/57 cases with the standard and modified method, respectively), although the sensitivity of the test was poor (23.5%). Here, we propose a practical and cost-effective approach combining the modified CDD and the CTX-M-MULTI test for detection of ESBL and/or AmpC in class A and B CPE. IMPORTANCE Antimicrobial resistance is a growing public health threat of broad concern worldwide. Timely detection of antibiotic resistance mechanisms can help to monitor and to curb the spread of resistant bacteria within the hospital setting as well as in the environment. In this work we report an accurate and affordable method to phenotypically identify difficult-to-detect resistance determinants in highly resistant (carbapenemase-producing) bacteria. This method may be implemented in any diagnostic microbiology lab and may reduce the underreporting of relevant resistance mechanisms.

Concerning Increase in Antimicrobial Resistance Patterns of Pathogenic Strains of Salmonella Isolated in Poultry Meat Products.

Salmonella is considered to be one of the major foodborne pathogens associated with the consumption of contaminated poultry meat products. To the best of our knowledge this is the first extended research performed on a number of Salmonella strains isolated during 2011-2021 from poultry meat products in Romania. The aim of this study was to characterize the prevalence of pathogenic Salmonella serovars, antimicrobial susceptibility, and antimicrobial resistance genes in 112 Salmonella isolates recovered from raw poultry meat products. The results showed that Salmonella enterica serovars Enteritidis and Typhimurium were the common serotypes (56%; 25%). Overall, the majority of the isolates were resistant to at least three tested antimicrobials. High resistance was observed for tetracycline (84%), nalidixic acid (78%), and ampicillin (78%) in pathogenic Salmonella isolated during the period 2016-2021. All the pathogenic Salmonella isolated during 2016-2021 tested positive to at least one resistance gene encoding for tetracycline resistance, with the tetA gene being the most prevalent (62%). In addition, 64% (24/37) of the Salmonella isolates carried at least one of the genes (blaCMY-2, blaSHV1, blaTEM1) that code for β-Lactams resistance. The findings in this study showed a high prevalence of multi-drug resistant (MDR) Salmonella serovars in poultry meat products and a concerning increase of resistance patterns. The continuous occurrence of more resistant strains implies that effective measures should be strictly applied in this particular food chain in order to prevent their spread and guarantee microbial safety.

Multi-drug and β-lactam resistance in Escherichia coli and food-borne pathogens

Abstract Antimicrobial resistance (AMR) is one of the top ten global public health threats facing humanity by menacing the effective use of available antimicrobials for treatment of an ever-increasing range of infections caused by bacteria. Its potential recognize the transmission to humans by contact with commensal and zoonotic resistant bacteria from animal and food sources. The aim of this study is to describe multi-drug resistance (MDR) profile in Campylobacter (n = 541), Salmonella (n = 919) and commensal Escherichia coli (n = 2777) isolated from animal and food samples between 2014 and 2019 in Portugal. Antimicrobial susceptibility testing results to fluoroquinolones/quinolones, macrolides, 3rd generation cephalosporins, polymyxins, carbapenems, penicillins, aminoglycosides, tetracyclines, sulphonamides, trimethoprim and chloramphenicol were clustered using k-modes. Clusters were described by population (broilers, broilers meat, turkeys, pigs, pig meat), AMR classification (mono/dual-resistance, MDR to 3-4, 5-6 and ≥7), β-lactamases production, sample stage (farm, slaughterhouse, processing plant, retail), season, and year. Overall, the results suggest an escalating MDR behavior from farm to post-farm stages in all bacteria, including E. coli MDR to critically important antimicrobials, and that Salmonella (fluoro)quinolones resistance may be associated with broilers. Most ESBL/AmpC producing E. coli were resistant to both C3G and C4G in isolates from animal and food samples (respectively, 77%; n = 846; and 74%; n = 188). The statistical method, K-modes, offers an overall visualization of the data with the identification of AMR profiles that have been further described using surveillance variables. Our results provide relevant information to support policy and decision making to tackle MDR in farm and post-farm stages. Key messages • An escalating multi drug resistance behavior from farm to post-farm stages. • Escherichia coli multi drug resistance to critically important antimicrobials.

Study of antibiotic resistance in freshwater ecosystems with low anthropogenic impact

This study aimed to investigate the bacterial diversity and the background level of antibiotic resistance in two freshwater ecosystems with low anthropogenic impact in order to evaluate the presence of natural antimicrobial resistance in these areas and its potential to spread downstream. Water samples from a pre-Alpine and an Apennine river (Variola and Tiber, respectively) were collected in three different sampling campaigns and bacterial diversity was assessed by 16S sequencing, while the presence of bacteria resistant to five antibiotics was screened using a culturable approach.Overall bacterial load was higher in the Tiber River compared with the Variola River. Furthermore, the study revealed the presence of resistant bacteria, especially the Tiber River showed, for each sampling, the presence of resistance to all antibiotics tested, while for the Variola River, the detected resistance was variable, comprising two or more antibiotics. Screening of two resistance genes on a total of one hundred eighteen bacterial isolates from the two rivers showed that blaTEM, conferring resistance to β-lactam antibiotics, was dominant and present in ~58 % of isolates compared to only ~9 % for mefA/E conferring resistance to macrolides. Moreover, β-lactam resistance was detected in various isolates showing also resistance to additional antibiotics such as macrolides, aminoglycosides and tetracyclines. These observations would suggest the presence of co-resistant bacteria even in non-anthropogenic environments and this resistance may spread from the environment to humans and/or animals.