Elucidation of the Gemcitabine Transporters of Escherichia coli K-12 and Gamma-Proteobacteria Linked to Gemcitabine-Related Chemoresistance.

Gemcitabine (2',2'-difluoro-2'-deoxycytidine), a widely used anticancer drug, is considered a gold standard in treating aggressive pancreatic cancers. Gamma-proteobacteria that colonize the pancreatic tumors contribute to chemoresistance against gemcitabine by metabolizing the drug to a less active and deaminated form. The gemcitabine transporters of these bacteria are unknown to date. Furthermore, there is no complete knowledge of the gemcitabine transporters in Escherichia coli or any other related proteobacteria. In this study, we investigate the complement of gemcitabine transporters in E. coli K-12 and two common chemoresistance-related bacteria (Klebsiella pneumoniae and Citrobacter freundii). We found that E. coli K-12 has two high-affinity gemcitabine transporters with distinct specificity properties, namely, NupC and NupG, whereas the gemcitabine transporters of C. freundii and K. pneumoniae include the NupC and NupG orthologs, functionally indistinguishable from their counterparts, and, in K. pneumoniae, one additional NupC variant, designated KpNupC2. All these bacterial transporters have a higher affinity for gemcitabine than their human counterparts. The highest affinity (KM 2.5-3.0 µΜ) is exhibited by NupGs of the bacteria-specific nucleoside-H+ symporter (NHS) family followed by NupCs (KM 10-13 µΜ) of the concentrative nucleoside transporter (CNT) family, 15-100 times higher than the affinities reported for the human gemcitabine transporter hENT1/SLC29A1, which is primarily associated with gemcitabine uptake in the pancreatic adenocarcinoma cells. Our results offer a basis for further insight into the role of specific bacteria in drug availability within tumors and for understanding the structure-function differences of bacterial and human drug transporters.

Multiplex lateral flow test strip for detection of carbapenemase genes using barcoded tetrahedral DNA capture probe-based biosensing interface.

Carbapenem-resistant Enterobacterales pose significant global health challenges due to their rapid spread and ability to hydrolyse various beta-lactam antibiotics. Rapid tests for these carbapenemase genes are crucial to ensure appropriate prescription administration and infection control. In this study, we developed a rapid visual nanodiagnostic platform for multiplexed detection of carbapenemase genes using a lateral flow strip. The nanodiagnostic strip was designed with separate barcoded DNA tetrahedrons for the blaKPC and blaNDM genes. These tetrahedrons were distributed on a nitrocellulose membrane at two different test lines as capture probes. When tested against a panel of carbapenemase genes, the tetrahedral probes captured single-stranded amplicons of asymmetric PCR via strand hybridisation. The amplicons acted as bridging elements, binding the DNA-modified gold nanoparticles to the test line of the strip, resulting in clear visual readouts specific to the blaKPC and blaNDM genes. By employing barcoded tetrahedrons and asymmetric PCR in conjunction with the lateral flow strip, a single diagnostic test enabled the detection of multiple carbapenemase genes. The test yielded results as low as 0.12 fM for blaKPC and 0.05 fM for blaNDM within 75 min. Furthermore, the strip effectively identified specific carbapenemase genes in clinical isolates using real-time PCR, antibody-based lateral flow systems for carbapenemase detection, and carbapenemase phenotype experiments. Thus, the strip develop has a high potential for testing blaKPC and blaNDM genes in practice.

Colonization of White-Tailed Deer (Odocoileus virginianus) from Urban and Suburban Environments with Cephalosporinase- and Carbapenemase-Producing Enterobacterales.

Wildlife play a role in the acquisition, maintenance, and dissemination of antimicrobial resistance (AMR). This is especially true at the human-domestic animal-wildlife interface, like urbanized areas, where interactions occur that can promote the cross-over of AMR bacteria and genes. We conducted a 2-year fecal surveillance (n = 783) of a white-tailed deer (WTD) herd from an urban park system in Ohio to identify and characterize cephalosporin-resistant and carbapenemase-producing bacteria using selective enrichment. Using generalized linear mixed models we found that older (OR = 2.3, P < 0.001), male (OR = 1.8, P = 0.001) deer from urbanized habitats (OR = 1.4, P = 0.001) were more likely to harbor extended-spectrum cephalosporin-resistant Enterobacterales. In addition, we isolated two carbapenemase-producing Enterobacterales (CPE), a Klebsiella quasipneumoniae harboring blaKPC-2 and an Escherichia coli harboring blaNDM-5, from two deer from urban habitats. The genetic landscape of the plasmid carrying blaKPC-2 was unique, not clustering with other reported plasmids encoding KPC-2, and only sharing 78% of its sequence with its nearest match. While the plasmid carrying blaNDM-5 shared sequence similarity with other reported plasmids encoding NDM-5, the intact IS26 mobile genetic elements surrounding multiple drug resistant regions, including the blaNDM-5, has been reported infrequently. Both carbapenemase genes were successfully conjugated to a J53 recipient conferring a carbapenem-resistant phenotype. Our findings highlight that urban environments play a significant role on the transmission of AMR bacteria and genes to wildlife and suggest WTD may play a role in the dissemination of clinically and epidemiologically relevant antimicrobial resistant bacteria. IMPORTANCE The role of wildlife in the spread of antimicrobial resistance is not fully characterized. Some wildlife, including white-tailed deer (WTD), can thrive in suburban and urban environments. This may result in the exchange of antimicrobial resistant bacteria and resistance genes between humans and wildlife, and lead to their spread in the environment. We found that WTD living in an urban park system carried antimicrobial resistant bacteria that were important to human health and resistant to antibiotics used to treat serious bacterial infections. This included two deer that carried bacteria resistant to carbapenem antibiotics which are critically important for treatment of life-threatening infections. These two bacteria had the ability to transfer their AMR resistance genes to other bacteria, making them a threat to public health. Our results suggest that WTD may contribute to the spread of antimicrobial resistant bacteria in the environment.

Isolation of a Colistin-Susceptible MDR Pantoea calida Harboring the mcr-9 Gene Suggests the Silent Spread of the Resistance Factor.

Pantoea spp. are bacteria that are often detected in the environment and as symbionts of arthropods. They sporadically cause infections in humans and recently extended spectrum beta-lactamases (ESBL)- and carbapenemase-producing strains have started to emerge. In this study, we report the isolation and the complete genome sequence of a strain of Pantoea calida encoding the colistin-resistance gene mcr-9. The strain was isolated from a preterm newborn in a neonatal pathology ward. On clinical examination, his vital signs were normal and blood culture was negative. Rectal swab screening for ESBL-producing Enterobacterales allowed to isolate the bacterium, and a complete genome was obtained using both short and long read sequencing. The mcr-9 gene was found to be encoded on a IncHI2 superplasmid, which confers resistance to six classes of antibiotics, including beta lactams (ESBL). Despite the presence of mcr-9, the isolate retains susceptibility to colistin, which could be explained by the absence of compatible regulatory genes (qseBC) from the genome. The presence of the resistance gene is undetectable with the routine clinical procedures, that is, phenotypic tests. This suggests that a silent spread might be ongoing in the ward. To our knowledge, this is the first description of an MDR P. calida and of a Pantoea spp. encoding any mobile colistin resistance gene.

Evaluating NG-Test CARBA 5 Multiplex Immunochromatographic and Cepheid Xpert CARBA-R Assays among Carbapenem-Resistant Enterobacterales Isolates Associated with Bloodstream Infection.

Decreased susceptibility to carbapenems in Enterobacterales is an emerging concern. Conventional methods with short turnaround times are crucial for therapeutic decisions and infection control. In the current study, we used the Xpert CARBA-R (Cepheid, Sunnyvale, CA, USA) and the NG-Test CARBA 5 (NG Biotech, Guipry, France) assays for carbapenemase detection in 214 carbapenem-resistant Enterobacterales (CRE) blood isolates. We used the modified carbapenem inactivation method, conventional PCR, and sequencing to determine the production of five common carbapenemase families and their subtypes. We performed wzc-genotyping for all CR-Klebsiella pneumoniae (CRKP) and multilocus sequence typing for all carbapenemase-producing CRE isolates to reveal their genetic relatedness. The results showed a sensitivity of 99.8% and a specificity of 100% by the Xpert assay, and a sensitivity of 100% and a specificity of 99% by the NG-Test in detecting carbapenemases of 84 CRKP isolates with only one (VIM-1+IMP-8) failure in both tests. For CR-Escherichia coli, four carbapenemase-producing isolates were detected accurately for their subtypes. The two major clones of carbapenemase-producing CRKP isolates in Taiwan were ST11-K47 producing KPC-2 (n = 47) and ST11-K64 producing OXA-48-like (n = 9). Our results support the use of either test in routine laboratories for the rapid detection of common carbapenemases. Caution should be taken using the Xpert assay in areas with a high prevalence of CRE carrying blaIMP-8. IMPORTANCE Carbapenemase-producing Enterobacterales (CPE) are emerging worldwide, causing nosocomial outbreaks and even community-acquired infections since their appearance 2 decades ago. Our previous national surveillance of CPE isolates in Taiwan identified five carbapenemase families (KPC, OXA, NDM, VIM, and IMP) with the KPC-2 and OXA-48-like types predominant. Timely detection and classification of carbapenemases in CPE may be a useful test to guide optimal therapy and infection control. Genetic detection methods using the Xpert CARBA-R assay and the immunochromatographic assay using the NG-Test CARBA 5 have been validated with the advantage of short turnaround time. Our study demonstrated that the NG and Xpert assays are convenient methods to accurately identify carbapenemases in carbapenem-resistant Klebsiella pneumoniae and carbapenem-resistant Escherichia coli blood isolates. Detecting IMP variants remains challenging, and the results of Xpert CARBA-R assay should be carefully interpreted.

Phenotype-genotype correlations among carbapenem-resistant Enterobacterales recovered from four Egyptian hospitals with the report of SPM carbapenemase.

BACKGROUND: Carbapenem-resistant Enterobacterales (CRE), currently listed by the World Health Organization (WHO) as top priority critical pathogens, are a major global menace to human health. In low- and middle-income countries (LMICs) the threat is mounting fueled by selective pressures caused by antibiotic abuse and inadequate diagnostic resources. METHODS: This study phenotypically and genotypically characterized carbapenem resistance among 115 Enterobacterales isolates including 76 Klebsiella (K.) pneumoniae, 19 Escherichia (E.) coli, 14 Shigella (S.) sonnei, 5 Enterobacter (E.) cloacae, and 1 Proteus (P.) mirabilis. RESULTS: Ninety-three isolates (80.9%) were carbapenem-resistant with an alarming 57.5% carbapenem non-susceptibility in isolates collected from the outpatient department. Molecular characterization of the carbapenemases (CPases) encoding genes showed that blaNDM (80.5%) was the most prevalent; it was detected in 62 isolates (54 K. pneumoniae, 6 E. coli and 2 S. sonnei), followed by blaVIM (36.4%) which was observed in 28 isolates (24 K. pneumoniae, 3 E. coli and 1 E. cloacae). Other CPases included blaKPC (28.6%; in 20 K. pneumoniae, 1 E. coli and 1 S. sonnei), blaOXA-48 (26%; in 17 K. pneumoniae, 1 E. coli,1 E. cloacae and 1 P. mirabilis), blaIMP (6.5%; in 5 K. pneumoniae) and blaSPM (1.3%; in K. pneumoniae). Notably more than half of the Enterobacterales isolates (54.5%) co-harboured more than one CPase-encoding gene. Co-existence of blaNDM and blaVIM genes was the most dominant (31.2%), followed by association of blaNDM and blaKPC (24.7%), then blaVIM and blaKPC (13%). Moreover, the effects of different genotypes on meropenem MIC values were assessed, and a statistically significant difference between the genotype (Ambler classes A and B) and the genotype (Ambler classes B and D) was recorded. CONCLUSION: The current findings may serve for a better understanding of the context of CRE in Egypt, associated drivers and CPases.

Evolution of ColE1-like plasmids across γ-Proteobacteria: From bacteriocin production to antimicrobial resistance.

Antimicrobial resistance is one of the major threats to Public Health worldwide. Understanding the transfer and maintenance of antimicrobial resistance genes mediated by mobile genetic elements is thus urgent. In this work, we focus on the ColE1-like plasmid family, whose distinctive replication and multicopy nature has given rise to key discoveries and tools in molecular biology. Despite being massively used, the hosts, functions, and evolutionary history of these plasmids remain poorly known. Here, we built specific Hidden Markov Model (HMM) profiles to search ColE1 replicons within genomes. We identified 1,035 ColE1 plasmids in five Orders of γ-Proteobacteria, several of which are described here for the first time. The phylogenetic analysis of these replicons and their characteristic MOBP5/HEN relaxases suggest that ColE1 plasmids have diverged apart, with little transfer across orders, but frequent transfer across families. Additionally, ColE1 plasmids show a functional shift over the last decades, losing their characteristic bacteriocin production while gaining several antimicrobial resistance genes, mainly enzymatic determinants and including several extended-spectrum betalactamases and carbapenemases. Furthermore, ColE1 plasmids facilitate the intragenomic mobilization of these determinants, as various replicons were identified co-integrated with large non-ColE1 plasmids, mostly via transposases. These results illustrate how families of plasmids evolve and adapt their gene repertoires to bacterial adaptive requirements.

Evaluation of the new BL-RED (ß-Lactamase Rapid Electrochemical Detection) test in positive blood culture broths.

The rapid detection of extended-spectrum ß-lactamase Enterobacterales (ESBL-E) in a positive blood culture is important in order to initiate an appropriate antibiotic therapy and thus decrease mortality. We evaluated the new BL-RED (ß-Lactamase Rapid Electrochemical Detection) test in 100 positive blood culture broths to detect (in ten minutes) the presence or absence of ESBL-E. The BL-RED test appears to be an easy, rapid and reliable test to detect the presence of ESBL directly in Gram negative bacilli-positive blood culture broths, with good performances (sensibility =97.3%, specificity =90.5%, predictive positive value =85.7% and predictive negative value =98.3%). This test could be useful for therapeutic decisions and adjustments of sepsis empirical antibiotic therapy, particularly in wards where the ecology is unfavorable, such as in intensive care units.

Trends of major antimicrobial resistance phenotypes in enterobacterales and gram-negative non-fermenters from ATLAS and EARS-net surveillance systems: Italian vs. European and global data, 2008-2018.

Antimicrobial resistance (AMR) is a growing health concern over the recent years. High AMR levels have been reported in Italy among European countries. Here, we analyze longitudinally the AMR trends observed in Italy for Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Enterobacter cloacae and Pseudomonas aeruginosa from the Antimicrobial Testing Leadership and Surveillance database, in comparison with data from the European Antimicrobial Resistance Surveillance Network (2008-2018). We also compare these longitudinal data from Italy with those from Europe and globally. Data analysis revealed highest resistance rates for carbapenems and difficult-to-treat resistance in A. baumannii (82.4% and 83.6%, respectively) followed by third-generation cephalosporin-resistant K. pneumoniae in Italy (≥50%). Resistance rates in Italy were higher compared to Europe and globally, as observed in both Antimicrobial Testing Leadership and Surveillance and European Antimicrobial Resistance Surveillance Network. These findings further substantiate the high AMR rates in Italy and aim to support informed decision making at a national level.

Imipenem-Relebactam Susceptibility and Genotypic Characteristics of Carbapenem-Resistant Enterobacterales Identified during Population-Based Surveillance.

Laboratories submit all carbapenem-resistant Enterobacter, Escherichia coli, and Klebsiella species to the Alameda County Public Health Department (ACPHD). ACPHD evaluated 75 isolates submitted during 9 months for susceptibility to imipenem-relebactam (I-R) and, using whole-genome sequencing, identified ß-lactamase genes. Of 60 (80%) isolates susceptible to I-R, 8 (13%) had detectable carbapenemase genes, including 4 KPC, two NDM, and two OXA-48-like; we described the relationship between the presence of ß-lactamase resistance genes and susceptibility to I-R.

Shipworm symbiosis ecology-guided discovery of an antibiotic that kills colistin-resistant Acinetobacter.

Teredinibacter turnerae is an intracellular bacterial symbiont in the gills of wood-eating shipworms, where it is proposed to use antibiotics to defend itself and its animal host. Several biosynthetic gene clusters are conserved in T. turnerae and their host shipworms around the world, implying that they encode defensive compounds. Here, we describe turnercyclamycins, lipopeptide antibiotics encoded in the genomes of all sequenced T. turnerae strains. Turnercyclamycins are bactericidal against challenging Gram-negative pathogens, including colistin-resistant Acinetobacter baumannii. Phenotypic screening identified the outer membrane as the likely target. Turnercyclamycins and colistin operate by similar cellular, although not necessarily molecular, mechanisms, but turnercyclamycins kill colistin-resistant A. baumannii, potentially filling an urgent clinical need. Thus, by exploring environments that select for the properties we require, we harvested the fruits of evolution to discover compounds with potential to target unmet health needs. Investigating the symbionts of shipworms is a powerful example of this principle.

Identification of Nasal Gammaproteobacteria with Potent Activity against Staphylococcus aureus: Novel Insights into the "Noncarrier" State.

Staphylococcus aureus nasal carriage provides the bacterial reservoir for opportunistic infection. In comparing the nasal microbiomes of culture-defined persistent S. aureus carriers versus noncarriers, we detected S. aureus DNA in all noses, including those with an established history of S. aureus negativity based on culture. Colonization with Gammaproteobacteria, including Klebsiella aerogenes, Citrobacter koseri, Moraxella lincolnii, and select Acinetobacter spp., was associated with S. aureus noncarriage. We next developed physiological competition assays for testing anti-S. aureus activity of isolated nasal species, utilizing medium modeling the nutrient-limited fluid of the nasal mucosa, polarized primary nasal epithelia, and nasal secretions. K. aerogenes from the nose of an S. aureus noncarrier demonstrated >99% inhibition of S. aureus recovery in all assays, even when S. aureus was coincubated in 9-fold excess. Secreted S. aureus inhibitory proteins from K. aerogenes and M. lincolnii were heat-stable and <30 kDa, fitting the profile of antimicrobial peptides. C. koseri, Acinetobacter haemolyticus, Acinetobacter junii, and Acinetobacter schindleri inhibited S. aureus recovery on nasal epithelia in a contact-dependent manner, while several other species either had no effect or promoted S. aureus growth. Collectively, this project is one of the first to identify resident nasal microbial species that impede S. aureus survival, and it implies that detectable nasal S. aureus results from shifts in microbial community composition.IMPORTANCE Nasal carriage of Staphylococcus aureus is a risk factor for infection, but it is not yet understood why some individuals carry nasal S. aureus persistently, intermittently, or seemingly not at all when tested via culture methods. This study compared the nasal microbiomes of established S. aureus carriers and noncarriers, identified species associated with noncarriage, and tested them for anti-S. aureus activity using assays developed to model the nutrient-limited nasal mucosa. We determined that all nostril swabs contain S. aureus DNA, even swabs from hosts considered to be long-term noncarriers. Select members of the Gammaproteobacteria class were more prevalent in noncarrier than carrier nostrils and demonstrated potent activity against multiple strains of S. aureus The results described here provide a better understanding of how the nasal microbiome controls S. aureus growth and viability and may be useful in the design of improved S. aureus decolonization strategies.

Diversity and functional analysis of Chinese bumblebee gut microbiota reveal the metabolic niche and antibiotic resistance variation of Gilliamella.

Bumblebees play an important role in maintaining the balance of natural and agricultural ecosystems, and the characteristic gut microbiota of bumblebees exhibit significant mutualistic functions. China has the highest diversity of bumblebees; however, gut microbiota of Chinese bumblebees have mostly been investigated through culture-independent studies. Here, we analyzed the gut communities of bumblebees from Sichuan, Yunnan, and Shaanxi provinces in China through 16S ribosomal RNA amplicon sequencing and bacterial isolation. It revealed that the bumblebees examined in this study harbored two gut enterotypes as previously reported: one is dominated by Gilliamella and Snodgrassella, and the other is distinguished by prevalent environmental species. The gut compositions obviously varied among different individual bees. We then isolated 325 bacterial strains and the comparative genomic analysis of Gilliamella strains revealed that galactose and pectin digestion pathways were conserved in strains from bumblebees, while genes for the utilization of arabinose, mannose, xylose, and rhamnose were mostly lost. Only two strains from the Chinese bumblebees possess the multidrug-resistant gene emrB, which is phylogenetically closely related to that from the symbionts of soil entomopathogenic nematode. In contrast, tetracycline-resistant genes were uniquely present in three strains from the USA. Our results illustrate the prevalence of strain-level variations in the metabolic potentials and the distributions of antibiotic-resistant genes in Chinese bumblebee gut bacteria.

A Multicentered Study of the Clinical and Molecular Epidemiology of TEM- and SHV-type Extended-Spectrum Beta-Lactamase Producing Enterobacterales Infections in Children.

BACKGROUND: Extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales-(Ent) infections are increasing in pediatrics. Before CTX-M ESBL emerged, the most common infection-associated ESBL genes were TEM and SHV-type ESBLs. We sought to define the current epidemiology of Ent infections in children due to blaTEM and blaSHV (TEM-SHV-Ent). METHODS: A retrospective case-control analysis of children with TEM-SHV-Ent infections at 3 Chicago-area hospitals was performed. Cases had extended-spectrum-cephalosporin (ESC)-resistant infections due to blaTEM or blaSHV. DNA analysis assessed ß-lactamase (bla) genes, multilocus sequence types, and E. coli phylogenetic grouping. Controls had ESC-susceptible Ent infections, matched 3:1 to cases by age, source, and hospital. Clinical-epidemiologic infection predictors were assessed. RESULTS: Of 356 ESC-R-Ent isolates from children (median 4.3 years), 38 (10.7%) were positive solely for blaTEM-ESBL (26%) or blaSHV-ESBL genes (74%). Predominant organisms were Klebsiella (34.2%) and E. coli (31.6%); 67% of E. coli were phylogroup B2. Multilocus sequence types revealed multiple strains, 58% resistant to ≥3 antibiotic classes. On multivariable analysis, children with TEM-SHV-Ent infections more often had recent inpatient care (OR, 8.2), yet were diagnosed mostly as outpatients (OR, 25.6) and less in Neonatal Intensive Care Units (OR, 0.036) than controls. TEM-SHV-Ent patients had more gastrointestinal (OR, 23.7) and renal comorbidities (OR, 4.2). Differences in demographics, antibiotic exposure, and foreign bodies were not found. CONCLUSION: TEM-SHV-Ent are commonly linked to inpatient exposures in children with chronic conditions but most often present in outpatient settings. Clinicians should be aware of the potential increased risk for TEM-SHV-Ent infections in outpatients with gastrointestinal and renal comorbidities and histories of prolonged hospital stays.

Distribution and antimicrobial resistance profiles of bacterial species in stray dogs, hospital-admitted dogs, and veterinary staff in South Korea.

Transferring antimicrobial-resistant bacteria from companion animals to human hosts has become increasingly common. Data regarding antimicrobial susceptibility could help veterinarians to select the most appropriate antibiotic treatment. However, standardized and ongoing surveys regarding antimicrobial resistance remain limited. In this study, we investigated the antimicrobial-susceptibility patterns and trends of bacteria isolated from stray dogs, hospital-admitted dogs, and veterinary staff in South Korea from 2018 to 2019. The minimum inhibitory concentrations of different antimicrobials for Staphylococcus spp., Enterobacterales, and Enterococcus spp. were determined to establish representatives of different antibiotic classes relevant for treatment or surveillance. For coagulase-positive and -negative Staphylococci, resistance to gentamicin was <27 %, while that to ampicillin and penicillin was high (33-80 %). The mecA-detection rates among staphylococcal isolates were 28.5 %, 42.6 %, and 32 % from stray dogs, hospital-admitted dogs, and veterinary staffs, respectively. For Enterobacterales, resistance to carbapenems was low (0-6%). A total of 31.2 % and 18.9 % of Enterobacterales isolates from stray dogs and hospital-admitted dogs were confirmed to possess at least one of blaCTX-M, blaSHV, or blaTEM. Additionally, Enterococcus spp. isolates showed no resistance to vancomycin. These results demonstrate that dogs are commonly colonized with antimicrobial-resistant bacteria and highlight the need for further investigation.

Microbial diversity and co-occurrence patterns in deep soils contaminated by polycyclic aromatic hydrocarbons (PAHs).

Numerous studies have enriched our knowledge of the microbial community composition and metabolic versatility of contaminated soil. However, there remains a substantial gap regarding the bioassembly patterns of the indigenous microbial community distribution in contaminated deep soils. Herein, the indigenous microbial community structure diversity, function, and co-occurrence relationships in aged PAH-contaminated deep soil collected from an abandoned chemical facility were investigated using high-throughput sequencing. The results showed that the dominant phyla in all samples were responsible for PAH degradation and included Proteobacteria (20.86%-81.37%), Chloroflexi (2.03%-28.44%), Firmicutes (3.06%-31.16%), Actinobacteria (2.92%-11.91%), Acidobacteria (0.41%-12.68%), and Nitrospirae (0.81%-9.21%). Eighty biomarkers were obtained by linear discriminant analysis of effect size (LEfSe), and most of these biomarkers were PAH degraders. Functional predictions using Tax4Fun indicated that the aged contaminated soil has the potential for PAH degradation. Statistical analysis showed that in contrast with the PAH concentration, edaphic properties (nutrients and pH) were significantly correlated (r > 0.25, P < 0.01) with the bacterial community and functional composition. Co-occurrence network analysis (modularity index of 0.781) revealed non-random assembly patterns of the bacterial communities in the PAH-contaminated soils. The modules in the network were mainly involved in carbon and nitrogen cycles, organic substance degradation, and biological electron transfer processes. Microbes from the same module had strong ecological linkages. Additionally, SAR202 clade, Thermoanaerobaculum, Nitrospira, and Xanthomonadales, which were identified as keystone species, played an irreplaceable role in the network. Overall, our results suggested that environmental factors such as nutrients and pH, together with ecological function, are the main factors driving the assembly of microbial communities in aged PAH-contaminated deep soils.

Temporal distribution of microbial community in an industrial wastewater treatment system following crash and during recovery periods.

Water and soil contamination by industrial wastes is a global concern. Biological treatment of industrial wastewater using bioreactors allows the removal of organic matter and nutrients and enables either reuse or safe discharge. Wastewater bioremediation depends in part on the microbial communities present in the bioreactor. To ascertain which communities may play a role in the remediation process, the present study investigates the microbial community structure and diversity of microorganisms found in a full-scale membrane bioreactor (MBR) for industrial wastewater treatment. The study was carried out using high-throughput data observations following a failure (crash) of the MBR and during the extended recovery of the process. Results revealed a positive correlation between the MBR's ability to remove organic matter and its microbial community richness. The significant changes in relative microbial abundance between crash and recovery periods of the MBR revealed the important role of specific bacterial genera in wastewater treatment processes. A whole-genome metagenomics based comparison showed a clear difference in microbial makeup between two functional periods of MBR activity. The crash period was characterized by abundance in bacteria belonging to Achromobacter, Acinetobacter, Halomonas, Pseudomonas and an uncultured MBAE14. The recovery period on the other hand was characterized by Aquamicrobium and by Wenzhouxiangella marina. Our study also revealed some interesting functional pathways characterizing the microbial communities from the two periods of bioreactor function, such as Nitrate and Sulfate reduction pathways. These differences indicate the connection between the bacterial diversity of the MBR and its efficiency to remove TOC.

Response of particle-associated bacteria to long-term heavy metal contamination in a tropical estuary.

Estuaries being the connecting link between terrestrial and marine environment, experience spatial variations in the hydrographic variables as well as concentrations of pollutants. The present study reports a contrasting difference in the metal tolerance and enzyme activity of particle-associated bacteria (PAB) isolated from the upstream and downstream reaches of a tropical estuary [Cochin Estuary (CE) in the southwest coast of India], exposed to different levels of heavy metal contamination. The upstream of the estuary has been overloaded with heavy metals in the last few decades, while the downstream is less polluted. There were only 25% of culturable PAB phylogenetically common in both upstream and downstream. The PAB isolated from the upstream were dominated by γ-proteobacteria (48.1%) followed by α-proteobacteria (25.0%), while it was in the reverse order of α-proteobacteria (45.9%) and γ-proteobacteria (36.1%) in the downstream. More number of PAB from the upstream showed tolerance to higher concentrations of Zn and Cd. The Acinetobacter sp. MMRF1051 isolated from the upstream showed tolerance up to 250 mM Zn, 100 mM Cd, and 250 mM Ni. The enzyme expression profile of PAB from downstream was in the order of lipase > phosphatase > ß-glucosidase > aminopeptidase, while it was in the order of ß-glucosidase > lipase > aminopeptidase > phosphatase in the upstream of the estuary. The present study shows the selective pressure exerted by heavy metal pollution on the diversity of culturable bacteria associated with particulate matter in a tropical estuary. Also, the variation in their enzyme activities may impinge the remineralization of particulate organic matter (POM) in the system and may impart adverse impacts on ecosystem functioning.

Activity of temocillin against ESBL-, AmpC-, and/or KPC-producing Enterobacterales isolated in Poland.

We evaluated the in vitro effectiveness of temocillin and several commonly used antimicrobials against Enterobacterales bacteria in isolates from Polish patients. We tested 400 isolates: 260 extended-spectrum ß-lactamase (ESBL)- and/or ampC ß-lactamase (AmpC)-producing isolates; 40 Klebsiella pneumoniae carbapenemase (KPC)-producing isolates; and 100 ESBL-, AmpC-, and KPC-negative isolates. The minimal inhibitory concentrations (MICs) of temocillin and 16 other antimicrobials were determined by reference microdilution. We also determined the activities of fosfomycin and ceftazidime/avibactam in KPC-producing isolates. The antibiotic sensitivities were interpreted according to EUCAST, BSAC, and CLSI criteria. Overall, 91% of the isolates were susceptible to temocillin using the urinary tract infection breakpoint (≤ 32 mg/L), and 61.8% were susceptible using the systemic infection breakpoint (≤ 8 mg/L). Meropenem and imipenem were the most active drugs (MIC50 values of 0.06 and 0.5 mg/L, respectively). Colistin and ertapenem (both MIC50 = 0.12 mg/L) were less active than meropenem or imipenem, but some strains were 77% susceptible to each of them. Among the KPC-producing isolates, 42.5% had MIC values of ≤ 32 mg/L (urinary tract infection breakpoint), but 100% were resistant to temocillin (systemic infection breakpoint). Ceftazidime/avibactam was active against 100% of the KPC-producing isolates, and fosfomycin was active against 40%. The empirical susceptibility rate observed among the urinary isolates suggests that temocillin may be considered as an alternative to carbapenems in the absence of KPC-producing bacteria. With regard to isolates from other sources, temocillin might be useful as a documented therapy agent or an empirical treatment in hospitals with a low prevalence of ESBL/AmpC-producing strains.

KPC-producing Enterobacterales with uncommon carbapenem susceptibility profile in Vitek 2 system.

KPC-producing K. pneumoniae is an endemic challenge. Seven KPC-producing Enterobacterales showed unusual carbapenems susceptibility profile. These strains were resistance at least one carbapenem and the ertapenem MIC was lower than imipenem and/or meropenem MICs using Vitek 2™ system (bioMerieux). When E-test™ and disk diffusion methods were performed the carbapenems showed susceptible results.