Detection of antibiotic resistant Enterobacterales in commercial raw pet food: a preliminary study.

Introduction: Raw diets have become popular in companion animal nutrition, but these diets may be contaminated with harmful bacteria because heat processing is not utilized to mitigate pathogens during the production process. We analyzed 24 commercially available frozen raw canine and feline diets for extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E). Methods: Samples were incubated in tryptic soy broth augmented with 50 µg/mL ampicillin to enrich for ESBL-E. ESBL-E were isolated using CHROMagar ESBL plates and isolate identification and antibiotic susceptibility testing were confirmed using the VITEK®2 instrument. Results: ESBL-E were isolated from 42% (10/24) of raw diets, with E. coli, Enterobacter cloacae complex and Klebsiella pneumoniae predominating. Most ESBL-E isolates (71%, 32/45) were multidrug-resistant. Direct plating of samples onto tryptic soy agar yielded bacterial counts >6 log10 for 2 samples from two different manufacturers. Conclusion: This preliminary study justifies further investigation into the potential contribution of raw diets to the dissemination of antibiotic resistant bacteria in companion animals and domestic living spaces.

Risk factors for colonization with extended-spectrum cephalosporin-resistant and carbapenem-resistant Enterobacterales among hospitalized patients in Guatemala: An Antibiotic Resistance in Communities and Hospitals (ARCH) study.

Objectives: The spread of extended-spectrum cephalosporin-resistant Enterobacterales (ESCrE) and carbapenem-resistant Enterobacterales (CRE) has resulted in increased morbidity, mortality, and health care costs worldwide. To identify the factors associated with ESCrE and CRE colonization within hospitals, we enrolled hospitalized patients at a regional hospital located in Guatemala. Methods: Stool samples were collected from randomly selected patients using a cross-sectional study design (March-September, 2021), and samples were tested for the presence of ESCrE and CRE. Hospital-based and household variables were examined for associations with ESCrE and CRE colonization using lasso regression models, clustered by ward (n = 21). Results: A total of 641 patients were enrolled, of whom complete data sets were available for 593. Colonization with ESCrE (72.3%, n = 429/593) was negatively associated with carbapenem administration (odds ratio [OR] 0.21, 95% confidence interval [CI] 0.11-0.42) and positively associated with ceftriaxone administration (OR 1.61, 95% CI 1.02-2.53), as was reported hospital admission within 30 days of the current hospitalization (OR 2.84, 95% CI 1.19-6.80). Colonization with CRE (34.6%, n = 205 of 593) was associated with carbapenem administration (OR 2.62, 95% CI 1.39-4.97), reported previous hospital admission within 30 days of current hospitalization (OR 2.58, 95% CI 1.17-5.72), hospitalization in wards with more patients (OR 1.05, 95% CI 1.02-1.08), hospitalization for ≥4 days (OR 3.07, 95% CI 1.72-5.46), and intubation (OR 2.51, 95% CI 1.13-5.59). No household-based variables were associated with ESCrE or CRE colonization in hospitalized patients. Conclusion: The hospital-based risk factors identified in this study are similar to what has been reported for risk of health care-associated infections, consistent with colonization being driven by hospital settings rather than community factors. This also suggests that colonization with ESCrE and CRE could be a useful metric to evaluate the efficacy of infection and prevention control programs in clinics and hospitals.

3D Printers in Hospitals: Bacterial Contamination of Common and Antimicrobial 3D-Printed Material.

COVID-19 has presented hospitals with unique challenges. A SHEA Research Network survey showed that 40% reported "limited" or worse levels of personal protective equipment (PPE), and 13% were self-producing PPE to address those deficits, including 3D-printed items. However, we do not know how efficiently, if at all, 3D-printed materials can be disinfected. Additionally, two filaments, PLACTIVE and BIOGUARD, claim to be antimicrobial; they use copper nanocomposites and silver ions to reduce bacterial populations. We assess how PLACTIVE and BIOGUARD may be contaminated and how well they reduce contamination, and how readily Polylactic Acid (PLA), a standard 3D-printed material, may be disinfected. 3D-printed materials, including PLACTIVE and BIOGUARD, are readily contaminated with bacteria that are common in hospitals and can sustain that contamination. Our findings reveal that the levels of contamination on PLACTIVE and BIOGUARD can vary under specific conditions such as layer height or bacterial contact time, sometimes surpassing or falling short of PLA. However, disinfected disks had lower overall CFU averages than those that were not, but the level of disinfection was variable, and bacterial populations recovered hours after disinfection application. Proper disinfection and using appropriate 3D-printed materials are essential to limit bacterial contamination. 3D printers and their products can be invaluable for hospitals, especially when supplies are low, and healthcare worker safety is paramount. Environmental services should be made aware of the presence of antimicrobial 3D-printed materials, and patients should be discouraged from printing their own items for use in hospital environments.

Characterization of diarrheagenic Escherichia coli from different cattle production systems in Brazil.

Diarrheagenic E. coli (DEC) can cause severe diarrhea and is a public health concern worldwide. Cattle are an important reservoir for this group of pathogens, and once introduced into the abattoir environment, these microorganisms can contaminate consumer products. This study aimed to characterize the distribution of DEC [Shiga toxin-producing E. coli (STEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), and enteroaggregative E. coli (EAEC)] from extensive and intensive cattle production systems in Brazil. Samples (n = 919) were collected from animal feces (n = 200), carcasses (n = 600), meat cuts (n = 90), employee feces (n = 9), and slaughterhouse water (n = 20). Virulence genes were detected by PCR in 10% of animal samples (94/919), with STEC (n = 81) as the higher prevalence, followed by EIEC (n = 8), and lastly EPEC (n = 5). Animals raised in an extensive system had a higher prevalence of STEC (average 48%, sd = 2.04) when compared to animals raised in an intensive system (23%, sd = 1.95) (Chi-square test, P < 0.001). From these animals, most STEC isolates only harbored stx2 (58%), and 7% were STEC LEE-positive isolates that were further identified as O157:H7. This study provides further evidence that cattle are potential sources of DEC, especially STEC, and that potentially pathogenic E. coli isolates are widely distributed in feces and carcasses during the slaughter process.

Persistence of heterologous Flavobacterium psychrophilum genetic variants in microcosms simulating fish farm and hatchery environments.

Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease, causes substantial economic losses in salmonid farms and hatcheries. Some multilocus sequence types (ST) of F. psychrophilum are more likely to be associated with fish farms and hatcheries, but it is unclear if these patterns of association represent genetic lineages that are more adapted to aquaculture environments. Towards elucidating the disease ecology of F. psychrophilum, the culturability of 10 distinct F. psychrophilum STs was evaluated for 13 weeks in three microcosms including sterilized well water, sterilized well water with commercial trout feed, or sterilized well water with raceway detritus. All STs remained culturable in each of the microcosms for at least 8 weeks, with bacterial concentrations often highest in the presence of raceway detritus. In addition, most (e.g., 90%) STs remained culturable for at least 13-weeks. Significant differences in log10 cfus were observed among STs, both within and between microcosms, suggesting potential variability in environmental persistence capacity among specific variants. Collectively, results highlight the ability of F. psychrophilum to not only persist for weeks under nutrient-limited conditions but also thrive in the presence of organic substrates common in fish farms and hatchery-rearing units.

ArcB orchestrates the quorum-sensing system to regulate type III secretion system 1 in Vibrio parahaemolyticus.

In many Vibrio species, virulence is regulated by quorum sensing, which is regulated by a complex, multichannel, two-component phosphorelay circuit. Through this circuit, sensor kinases transmit sensory information to the phosphotransferase LuxU via a phosphotransfer mechanism, which in turn transmits the signal to the response regulator LuxO. For Vibrio parahaemolyticus, type III secretion system 1 (T3SS1) is required for cytotoxicity, but it is unclear how quorum sensing regulates T3SS1 expression. Herein, we report that a hybrid histidine kinase, ArcB, instead of LuxU, and sensor kinase LuxQ and response regulator LuxO, collectively orchestrate T3SS1 expression in V. parahaemolyticus. Under high oxygen conditions, LuxQ can interact with ArcB directly and phosphorylates the Hpt domain of ArcB. The Hpt domain of ArcB phosphorylates the downstream response regulator LuxO instead of ArcA. LuxO then activates transcription of the T3SS1 gene cluster. Under hypoxic conditions, ArcB autophosphorylates and phosphorylates ArcA, whereas ArcA does not participate in regulating the expression of T3SS1. Our data provides evidence of an alternative regulatory path involving the quorum sensing phosphorelay and adds another layer of understanding about the environmental regulation of gene expression in V. parahaemolyticus.

Risk Factors for Colonization With Extended-Spectrum Cephalosporin-Resistant and Carbapenem-Resistant Enterobacterales Among Hospitalized Patients in Kenya: An Antibiotic Resistance in Communities and Hospitals (ARCH) Study.

BACKGROUND: The spread of extended-spectrum cephalosporin-resistant Enterobacterales (ESCrE) and carbapenem-resistant Enterobacterales (CRE) represents a significant global public health threat. We identified putative risk factors for ESCrE and CRE colonization among patients in 1 urban and 3 rural hospitals in Kenya. METHODS: During a January 2019 and March 2020 cross-sectional study, stool samples were collected from randomized inpatients and tested for ESCrE and CRE. The Vitek2 instrument was used for isolate confirmation and antibiotic susceptibility testing, and least absolute shrinkage and selection operator (LASSO) regression models were used to identify colonization risk factors while varying antibiotic use measures. RESULTS: Most (76%) of the 840 enrolled participants received ≥1 antibiotic in the 14 days preceding their enrollment, primarily ceftriaxone (46%), metronidazole (28%), or benzylpenicillin-gentamycin (23%). For LASSO models that included ceftriaxone administration, ESCrE colonization odds were higher among patients hospitalized for ≥3 days (odds ratio, 2.32 [95% confidence interval, 1.6-3.37]; P < .001), intubated patients (1.73 [1.03-2.91]; P = .009), and persons living with human immunodeficiency virus (1.70 [1.03-2.8]; P = .029). CRE colonization odds were higher among patients receiving ceftriaxone (odds ratio, 2.23 [95% confidence interval, 1.14-4.38]; P = .025) and for every additional day of antibiotic use (1.08 [1.03-1.13]; P = .002). CONCLUSIONS: While CRE colonization was strongly associated with ceftriaxone use and duration of antibiotic use, the odds of ESCrE colonization increased with exposure to the hospital setting and invasive medical devices, which may reflect nosocomial transmission. These data suggest several areas where hospitals can intervene to prevent colonization among hospitalized patients, both through robust infection prevention and control practices and antibiotic stewardship programs.

Colonization With Antibiotic-Resistant Bacteria in a Hospital and Associated Communities in Guatemala: An Antibiotic Resistance in Communities and Hospitals (ARCH) Study.

BACKGROUND: We estimated the prevalence of colonization with extended-spectrum cephalosporin-resistant Enterobacterales (ESCrE) and carbapenem-resistant Enterobacterales (CRE) from a hospital and associated communities in western Guatemala. METHODS: Randomly selected infants, children, and adults (<1, 1-17, and ≥18 years, respectively) were enrolled from the hospital (n = 641) during the coronavirus disease 2019 (COVID-19) pandemic, March to September 2021. Community participants were enrolled using a 3-stage cluster design between November 2019 and March 2020 (phase 1, n = 381) and between July 2020 and May 2021 (phase 2, with COVID-19 pandemic restrictions, n = 538). Stool samples were streaked onto selective chromogenic agar, and a Vitek 2 instrument was used to verify ESCrE or CRE classification. Prevalence estimates were weighted to account for sampling design. RESULTS: The prevalence of colonization with ESCrE and CRE was higher among hospital patients compared to community participants (ESCrE: 67% vs 46%, P < .01; CRE: 37% vs 1%, P < .01). Hospital ESCrE colonization was higher for adults (72%) compared with children (65%) and infants (60%) (P < .05). Colonization was higher for adults (50%) than children (40%) in the community (P < .05). There was no difference in ESCrE colonization between phase 1 and 2 (45% and 47%, respectively, P > .05), although reported use of antibiotics among households declined (23% and 7%, respectively, P < .001). CONCLUSIONS: While hospitals remain foci for ESCrE and CRE colonization, consistent with the need for infection control programs, community prevalence of ESCrE in this study was high, potentially adding to colonization pressure and transmission in healthcare settings. Better understanding of transmission dynamics and age-related factors is needed.

Risk Factors for Colonization With Multidrug-Resistant Bacteria in Urban and Rural Communities in Kenya: An Antimicrobial Resistance in Communities and Hospitals (ARCH) Study.

BACKGROUND: Colonization with antimicrobial-resistant bacteria increases the risk of drug-resistant infections. We identified risk factors potentially associated with human colonization with extended-spectrum cephalosporin-resistant Enterobacterales (ESCrE) in low-income urban and rural communities in Kenya. METHODS: Fecal specimens, demographic and socioeconomic data were collected cross-sectionally from clustered random samples of respondents in urban (Kibera, Nairobi County) and rural (Asembo, Siaya County) communities between January 2019 and March 2020. Presumptive ESCrE isolates were confirmed and tested for antibiotic susceptibility using the VITEK2 instrument. We used a path analytic model to identify potential risk factors for colonization with ESCrE. Only 1 participant was included per household to minimize household cluster effects. RESULTS: Stool samples from 1148 adults (aged ≥18 years) and 268 children (aged <5 years) were analyzed. The likelihood of colonization increased by 12% with increasing visits to hospitals and clinics. Furthermore, individuals who kept poultry were 57% more likely to be colonized with ESCrE than those who did not. Respondents' sex, age, use of improved toilet facilities, and residence in a rural or urban community were associated with healthcare contact patterns and/or poultry keeping and may indirectly affect ESCrE colonization. Prior antibiotic use was not significantly associated with ESCrE colonization in our analysis. CONCLUSIONS: The risk factors associated with ESCrE colonization in communities include healthcare- and community-related factors, indicating that efforts to control antimicrobial resistance in community settings must include community- and hospital-level interventions.

Conjugative transfer of streptococcal prophages harboring antibiotic resistance and virulence genes.

Prophages play important roles in the transduction of various functional traits, including virulence factors, but remain debatable in harboring and transmitting antimicrobial resistance genes (ARGs). Herein we characterize a prevalent family of prophages in Streptococcus, designated SMphages, which harbor twenty-five ARGs that collectively confer resistance to ten antimicrobial classes, including vanG-type vancomycin resistance locus and oxazolidinone resistance gene optrA. SMphages integrate into four chromosome attachment sites by utilizing three types of integration modules and undergo excision in response to phage induction. Moreover, we characterize four subtypes of Alp-related surface proteins within SMphages, the lethal effects of which are extensively validated in cell and animal models. SMphages transfer via high-frequency conjugation that is facilitated by integrative and conjugative elements from either donors or recipients. Our findings explain the widespread of SMphages and the rapid dissemination of ARGs observed in members of the Streptococcus genus.

High prevalence of intermediate resistance to ciprofloxacin in Salmonella enterica isolated from a Brazilian poultry production chain, located in Minas Gerais state.

We characterized the distribution and diversity of antimicrobial-resistance Salmonella enterica isolated from a poultry production chain in Minas Gerais, Brazil, with special attention to ciprofloxacin and multidrug resistance (MDR). S. enterica (n = 96) of different serotypes and from different processing steps were subjected to broth dilution assay to estimate the minimum inhibitory concentration (MIC) for 12 antibiotics (8 classes) and screened using PCR for the presence of 17 antimicrobial-resistance genes. Isolates presented mainly resistance to ampicillin (11/96), and most presented intermediate resistance to ciprofloxacin (92/96). Roughly one-third (33/96) were resistant to streptomycin based on our interpretive criteria. Most strains resistant to streptomycin and ciprofloxacin were PCR-positive for aphA (51/96) and qnrB (94/96), respectively. Ciprofloxacin resistance was further investigated through high-resolution melting qPCR (HRM-qPCR) and sequencing of quinolone resistance-determining region (QRDR: gyrA, gyrB, parC, and parE). Minor differences were identified in melting temperatures (Tm), and a Thr57Sr mutation was observed in parC. MDR isolates harboring acrA and capable of expressing the AcrAB-TolC multidrug efflux pump were resistant to ethidium bromide at 0.4 mg/mL. The intermediate resistance to ciprofloxacin may be associated with qnrB, and the potential role of Thr57Ser mutation warrants further investigation. The high prevalence of antibiotic related genes and its association with the observed intermediary resistance to ciprofloxacin indicates the widespread of this hazard in the studied poultry production chain.

Nitrogen Fixation and Ammonium Assimilation Pathway Expression of Geobacter sulfurreducens Changes in Response to the Anode Potential in Microbial Electrochemical Cells.

Nitrogen gas (N2) fixation in the anode-respiring bacterium Geobacter sulfurreducens occurs through complex, multistep processes. Optimizing ammonium (NH4+) production from this bacterium in microbial electrochemical technologies (METs) requires an understanding of how those processes are regulated in response to electrical driving forces. In this study, we quantified gene expression levels (via RNA sequencing) of G. sulfurreducens growing on anodes fixed at two different potentials (-0.15 V and +0.15 V versus standard hydrogen electrode). The anode potential had a significant impact on the expression levels of N2 fixation genes. At -0.15 V, the expression of nitrogenase genes, such as nifH, nifD, and nifK, significantly increased relative to that at +0.15 V, as well as genes associated with NH4+ uptake and transformation, such as glutamine and glutamate synthetases. Metabolite analysis confirmed that both of these organic compounds were present in significantly higher intracellular concentrations at -0.15 V. N2 fixation rates (estimated using the acetylene reduction assay and normalized to total protein) were significantly larger at -0.15 V. Genes expressing flavin-based electron bifurcation complexes, such as electron-transferring flavoproteins (EtfAB) and the NADH-dependent ferredoxin:NADP reductase (NfnAB), were also significantly upregulated at -0.15 V, suggesting that these mechanisms may be involved in N2 fixation at that potential. Our results show that in energy-constrained situations (i.e., low anode potential), the cells increase per-cell respiration and N2 fixation rates. We hypothesize that at -0.15 V, they increase N2 fixation activity to help maintain redox homeostasis, and they leverage electron bifurcation as a strategy to optimize energy generation and use. IMPORTANCE Biological nitrogen fixation coupled with ammonium recovery provides a sustainable alternative to the carbon-, water-, and energy-intensive Haber-Bosch process. Aerobic biological nitrogen fixation technologies are hindered by oxygen gas inhibition of the nitrogenase enzyme. Electrically driving biological nitrogen fixation in anaerobic microbial electrochemical technologies overcomes this challenge. Using Geobacter sulfurreducens as a model exoelectrogenic diazotroph, we show that the anode potential in microbial electrochemical technologies has a significant impact on nitrogen gas fixation rates, ammonium assimilation pathways, and expression of genes associated with nitrogen gas fixation. These findings have important implications for understanding regulatory pathways of nitrogen gas fixation and will help identify target genes and operational strategies to enhance ammonium production in microbial electrochemical technologies.

Perspective: Phosphorus monitoring must be rooted in sustainability frameworks spanning material scale to human scale.

Phosphorus (P) is a finite resource, and its environmental fate and transport is complex. With fertilizer prices expected to remain high for years and disruption to supply chains, there is a pressing need to recover and reuse P (primarily as fertilizer). Whether recovery is to occur from urban systems (e.g., human urine), agricultural soil (e.g., legacy P), or from contaminated surface waters, quantification of P in various forms is vital. Monitoring systems with embedded near real time decision support, so called cyber physical systems, are likely to play a major role in the management of P throughout agro-ecosystems. Data on P flow(s) connects the environmental, economic, and social pillars of the triple bottom line (TBL) sustainabilty framework. Emerging monitoring systems must account for complex interactions in the sample, and interface with a dynamic decision support system that considers adaptive dynamics to societal needs. It is known from decades of study that P is ubiquitous, yet without quantitative tools for studying the dynamic nature of P in the environment, the details may remain elusive. If new monitoring systems (including CPS and mobile sensors) are informed by sustainability frameworks, data-informed decision making may foster resource recovery and environmental stewardship from technology users to policymakers.

Prevalence of colonization with multidrug-resistant bacteria in communities and hospitals in Kenya.

We estimated the prevalence of extended-spectrum cephalosporin-resistant Enterobacterales (ESCrE), carbapenem-resistant Enterobacterales (CRE), and methicillin-resistant Staphylococcus aureus (MRSA) in communities and hospitals in Kenya to identify human colonization with multidrug-resistant bacteria. Nasal and fecal specimen were collected from inpatients and community residents in Nairobi (urban) and Siaya (rural) counties. Swabs were plated on chromogenic agar to presumptively identify ESCrE, CRE and MRSA isolates. Confirmatory identification and antibiotic susceptibility testing were done using the VITEK®2 instrument. A total of 1999 community residents and 1023 inpatients were enrolled between January 2019 and March 2020. ESCrE colonization was higher in urban than rural communities (52 vs. 45%; P = 0.013) and in urban than rural hospitals (70 vs. 63%; P = 0.032). Overall, ESCrE colonization was ~ 18% higher in hospitals than in corresponding communities. CRE colonization was higher in hospital than community settings (rural: 7 vs. 1%; urban: 17 vs. 1%; with non-overlapping 95% confidence intervals), while MRSA was rarely detected (≤ 3% overall). Human colonization with ESCrE and CRE was common, particularly in hospitals and urban settings. MRSA colonization was uncommon. Evaluation of risk factors and genetic mechanisms of resistance can guide prevention and control efforts tailored to different environments.

Estimating the population-level prevalence of antimicrobial-resistant enteric bacteria from latrine samples.

BACKGROUND: Logistical and economic barriers hamper community-level surveillance for antimicrobial-resistant bacteria in low-income countries. Latrines are commonly used in these settings and offer a low-cost source of surveillance samples. It is unclear, however, whether antimicrobial resistance prevalence estimates from latrine samples reflect estimates generated from randomly sampled people. METHODS: We compared the prevalence of antimicrobial-resistant enteric bacteria from stool samples of people residing in randomly selected households within Kibera-an informal urban settlement in Kenya-to estimates from latrine samples within the same community. Fecal samples were collected between November 2015 and Jan 2016. Presumptive Escherichia coli isolates were collected from each household stool sample (n = 24) and each latrine sample (n = 48), resulting in 8935 and 8210 isolates, respectively. Isolates were tested for resistance to nine antibiotics using the replica-plating technique. Correlation- and Kolmogorov-Smirnov (K-S) tests were used to compare results. RESULTS: Overall, the prevalence values obtained from latrine samples closely reflected those from stool samples, particularly for low-prevalence (< 15%) resistance phenotypes. Similarly, the distribution of resistance phenotypes was similar between latrine and household samples (r > 0.6; K-S p-values > 0.05). CONCLUSIONS: Although latrine samples did not perfectly estimate household antimicrobial resistance prevalence, they were highly correlated and thus could be employed as low-cost samples to monitor trends in antimicrobial resistance, detect the emergence of new resistance phenotypes and assess the impact of community interventions.

Excreted Antibiotics May Be Key to Emergence of Increasingly Efficient Antibiotic Resistance in Food Animal Production.

At a time when antibiotic resistance is seemingly ubiquitous worldwide, understanding the mechanisms responsible for successful emergence of new resistance genes may provide insights into the persistence and pathways of dissemination for antibiotic-resistant organisms in general. For example, Escherichia coli strains harboring a class A ß-lactamase-encoding gene (blaCTX-M-15) appear to be displacing strains that harbor a class C ß-lactamase gene (blaCMY-2) in Washington State dairy cattle. We cloned these genes with native promoters into low-copy-number plasmids that were then transformed into isogenic strains of E. coli, and growth curves were generated for two commonly administered antibiotics (ampicillin and ceftiofur). Both strains met the definition of resistance for ampicillin (≥32 µg/mL) and ceftiofur (≥16 µg/mL). Growth of the CMY-2-producing strain was compromised at 1,000 µg/mL ampicillin, whereas the CTX-M-15-producing strain was not inhibited in the presence of 3,000 µg/mL ampicillin or with most concentrations of ceftiofur, although there were mixed outcomes with ceftiofur metabolites. Consequently, in the absence of competing genes, E. coli harboring either gene would experience a selective advantage if exposed to these antibiotics. Successful emergence of CTX-M-15-producing strains where CMY-2-producing strains are already established, however, requires high concentrations of antibiotics that can only be found in the urine of treated animals (e.g., >2,000 µg/mL for ampicillin, based on literature). This ex vivo selection pressure may be important for the emergence of new and more efficient antibiotic resistance genes and likely for persistence of antibiotic-resistant bacteria in food animal populations. IMPORTANCE We studied the relative fitness benefits of a cephalosporin resistance enzyme (CTX-M-15) that is displacing a similar enzyme (CMY-2), which is extant in E. coli from dairy cattle in Washington State. In vitro experiments demonstrated that CTX-M-15 provides a significant fitness advantage, but only in the presence of very high concentrations of antibiotic that are only found when the antibiotic ampicillin, and to a lesser extent ceftiofur, is excreted in urine from treated animals. As such, the increasing prevalence of bacteria with blaCTX-M-15 is likely occurring ex vivo. Interventions should focus on controlling waste from treated animals and, when possible, selecting antibiotics that are less likely to impact the proximal environment of treated animals.

Point-prevalence survey of antibiotic use at three public referral hospitals in Kenya.

Antimicrobial stewardship encourages appropriate antibiotic use, the specific activities of which will vary by institutional context. We investigated regional variation in antibiotic use by surveying three regional public hospitals in Kenya. Hospital-level data for antimicrobial stewardship activities, infection prevention and control, and laboratory diagnostic capacities were collected from hospital administrators, heads of infection prevention and control units, and laboratory directors, respectively. Patient-level antibiotic use data were abstracted from medical records using a modified World Health Organization point-prevalence survey form. Altogether, 1,071 consenting patients were surveyed at Kenyatta National Hospital (KNH, n = 579), Coast Provincial General Hospital (CPGH, n = 229) and Moi Teaching and Referral Hospital (MTRH, n = 263). The majority (67%, 722/1071) were ≥18 years and 53% (563/1071) were female. Forty-six percent (46%, 489/1071) were receiving at least one antibiotic. Antibiotic use was higher among children <5 years (70%, 150/224) than among other age groups (40%, 339/847; P < 0.001). Critical care (82%, 14/17 patients) and pediatric wards (59%, 155/265) had the highest proportion of antibiotic users. Amoxicillin/clavulanate was the most frequently used antibiotic at KNH (17%, 64/383 antibiotic doses), and ceftriaxone was most used at CPGH (29%, 55/189) and MTRH (31%, 57/184). Forty-three percent (326/756) of all antibiotic prescriptions had at least one missed dose recorded. Forty-six percent (204/489) of patients on antibiotics had a specific infectious disease diagnosis, of which 18% (37/204) had soft-tissue infections, 17% (35/204) had clinical sepsis, 15% (31/204) had pneumonia, 13% (27/204) had central nervous system infections and 10% (20/204) had obstetric or gynecological infections. Of these, 27% (56/204) had bacterial culture tests ordered, with culture results available for 68% (38/56) of tests. Missed antibiotic doses, low use of specimen cultures to guide therapy, high rates of antibiotic use, particularly in the pediatric and surgical population, and preference for broad-spectrum antibiotics suggest antibiotic use in these tertiary care hospitals is not optimal. Antimicrobial stewardship programs, policies, and guidelines should be tailored to address these areas.

Characterization of Interactions between CTX-M-15 and Clavulanic Acid, Desfuroylceftiofur, Ceftiofur, Ampicillin, and Nitrocefin.

Cefotaximase-Munich (CTX-M) extended-spectrum beta-lactamases (ESBLs) are commonly associated with Gram-negative, hospital-acquired infections worldwide. Several beta-lactamase inhibitors, such as clavulanate, are used to inhibit the activity of these enzymes. To understand the mechanism of CTX-M-15 activity, we have determined the crystal structures of CTX-M-15 in complex with two specific classes of beta-lactam compounds, desfuroylceftiofur (DFC) and ampicillin, and an inhibitor, clavulanic acid. The crystal structures revealed that Ser70 and five other residues (Lys73, Tyr105, Glu166, Ser130, and Ser237) participate in catalysis and binding of those compounds. Based on analysis of steady-state kinetics, thermodynamic data, and molecular docking to both wild-type and S70A mutant structures, we determined that CTX-M-15 has a similar affinity for all beta-lactam compounds (ceftiofur, nitrocefin, DFC, and ampicillin), but with lower affinity for clavulanic acid. A catalytic mechanism for tested ß-lactams and two-step inhibition mechanism of clavulanic acid were proposed. CTX-M-15 showed a higher activity toward DFC and nitrocefin, but significantly lower activity toward ampicillin and ceftiofur. The interaction between CTX-M-15 and both ampicillin and ceftiofur displayed a higher entropic but lower enthalpic effect, compared with DFC and nitrocefin. DFC, a metabolite of ceftiofur, displayed lower entropy and higher enthalpy than ceftiofur. This finding suggests that compounds containing amine moiety (e.g., ampicillin) and the furfural moiety (e.g., ceftiofur) could hinder the hydrolytic activity of CTX-M-15.

Temporal Transcriptional Responses of a Vibrio alginolyticus Strain to Podoviridae Phage HH109 Revealed by RNA-Seq.

Phage are thought to exhibit control over host genes during infection. As a preliminary investigation of the kinetics and magnitude of co-expression between phage and bacteria, we compared the global transcriptional profiles for Vibrio alginolyticus strain E110 and its lytic phage HH109 by using RNA sequencing. In total, 24.7% (1,143/4,620) of the host protein-coding genes were differentially expressed genes during infection (DEGs). Functional analysis of the host DEGs suggests that phage HH109 induced rapid and distinctive changes when compared with 60- and 120-min postinfection (mpi). Based on gene co-expression network analysis, an uncharacterized late gene gp27 encoded by the phage HH109 was predicted to modulate the host's membrane transport and/or transcriptional regulation. Furthermore, expression of several bacterial virulence genes was downregulated while drug resistance genes were upregulated. This work contributes to an in-depth understanding of the reciprocal interactions of lytic phage HH109 and its pathogenic Vibrio host E110, and can provide new insights into the research and development of phage therapy against pathogenic Vibrio infections in the economically significant aquatic animals. IMPORTANCE Vibrio alginolyticus is a common opportunistic pathogen that causes mass mortality in cultured marine animals. Phage HH109 lyses pathogenic V. alginolyticus strain E110 with high efficiency and thus serves as a useful model to understand the dynamic interplay of a phage and its host. Global transcriptomic responses of strain E110 post-HH109 infection were characterized by using RNA sequencing, elucidating step-by-step control by HH109, an antiphage-like responses, and the elevated expression of drug resistance. This study provides a detailed molecular description phage and V. alginolyticus, providing insight into better prevention and control of vibriosis in aquatic animals.

Occurrence and Distribution of Antibiotic-Resistant Staphylococcus aureus in a Brazilian Pork Production Chain.

ABSTRACT: Here we characterized the distribution and the antibiotic resistance of staphylococci from a Brazilian pork production chain. Samples (n = 1,114) from pig farms, pig lots, and slaughterhouses, located in two Brazilian states (Minas Gerais and Paraná), were subjected to coagulase-positive Staphylococcus enumeration. S. aureus isolates (n = 251) from this collection were further characterized for their resistance to oxacillin, cefoxitin, vancomycin, and tetracycline through phenotypic and molecular assays. Coagulase-positive Staphylococcus counts from pig farms were higher compared with other samples (P < 0.05). Other counts were relatively low but were present in all production stages. S. aureus isolates were commonly resistant to oxacillin and cefoxitin (54 of 73, 74.0%), qualifying them as methicillin-resistant S. aureus, but PCR assays indicated that few harbored the expected antimicrobial resistance genes (femB, mecA, and mecC). Lower frequencies of vancomycin and tetracycline resistance were found (6.8 to 37.0%). PCR sensitivity (34.5 to 86.7%) and specificity (26.6 to 85.0%) for detection of antibiotic resistance genes varied based on the assessed antibiotic. Antibiotic-resistant staphylococci are widely distributed in the Brazilian pork production chain, and methicillin-resistant S. aureus can become a potential health and economic impediment for the Brazilian pork industry.