Selection of antibiotic-resistant bacterial populations in the dairy cow gut following intramuscular ceftiofur treatment for metritis.

Third-generation cephalosporins such as ceftiofur are critically important antibiotics because human pathogens with resistance to these drugs contribute to high mortality rates. These antibiotics are also frequently given to dairy cattle for treating infections, emphasizing the critical role they play in both human and veterinary medicine. To investigate the impact of intramuscular ceftiofur treatment on the concentration of resistant bacteria in the gut, we focused on cows with metritis, a common bacterial infection that frequently requires antibiotic intervention. Twelve cows with metritis (cases) were enrolled and treated with intramuscular ceftiofur for 5 d along with 12 matched healthy cows that were not given ceftiofur (controls). Fecal samples were collected weekly from cows in both the case and control groups for 4 weeks, starting before the treatment of the case group. Five fecal samples per cow were used for analysis (n = 120 samples). The abundance of Gram-negative bacteria was quantified per sample after plating on MacConkey agar, which was also used to quantify the abundance of Gram-negative bacteria with resistance to ceftiofur, ampicillin, and tetracycline. Interestingly, the case cows with metritis had a greater abundance of Gram-negative bacteria than the control cows just before treatment, but no difference in abundance was observed between groups at wk 1-4. The abundance of ceftiofur-resistant Gram-negative bacteria was also similar between the case and control cows immediately before treatment of the cases. However, a significant increase in abundance of ceftiofur-resistant Gram-negative bacteria was observed in the case cows 1-week after treatment that persisted through wk 3. Although the recovery of ampicillin- and tetracycline-resistant bacteria was similar between the 2 groups post-treatment, cases had significantly higher levels of ampicillin-resistant bacteria before treatment. Collectively, these findings demonstrate that intramuscular ceftiofur treatment can affect the abundance of cultivable Gram-negative bacteria and select for ceftiofur-resistant populations that can persist for up to 3 weeks. Judicious use practices are needed to ensure that ceftiofur and other critically important antibiotics are administered only when necessary to minimize the spread of resistance and safeguard public and animal health.

Anti-biofilm Activity of Human Milk Oligosaccharides in Clinical Strains of Streptococcus agalactiae with Diverse Capsular and Sequence Types.

Group B Streptococcus (GBS) is an encapsulated Gram-positive bacterial pathogen that causes severe perinatal infections. Human milk oligosaccharides (HMOs) are short-chain sugars that have recently been shown to possess antimicrobial and anti-biofilm activity against a variety of bacterial pathogens, including GBS. We have expanded these studies to demonstrate that HMOs can inhibit and dismantle biofilm in both invasive and colonizing strains of GBS. A cohort of 30 diverse strains of GBS were analyzed for susceptibility to HMO-dependent biofilm inhibition or destruction. HMOs were significantly effective at inhibiting biofilm in capsular-type- and sequence-type-specific fashion, with significant efficacy in CpsIb, CpsII, CpsIII, CpsV, and CpsVI strains as well as ST-1, ST-12, ST-19, and ST-23 strains. Interestingly, CpsIa as well as ST-7 and ST-17 were not susceptible to the anti-biofilm activity of HMOs, underscoring the strain-specific effects of these important antimicrobial molecules against the perinatal pathogen Streptococcus agalactiae.

Galacto-Oligosaccharide Supplementation Modulates Pathogen-Commensal Competition between Streptococcus agalactiae and Streptococcus salivarius.

The members of the infant microbiome are governed by feeding method (breastmilk vs. formula). Regardless of the source of nutrition, a competitive growth advantage can be provided to commensals through prebiotics - either human milk oligosaccharides (HMOs) or plant oligosaccharides that are supplemented into formula. To characterize how prebiotics modulate commensal - pathogen interactions, we have designed and studied a minimal microbiome where a pathogen, Streptococcus agalactiae engages with a commensal, Streptococcus salivarius. We discovered that while S. agalactiae suppresses the growth of S. salivarius via increased lactic acid production, galacto-oligosaccharides (GOS) supplementation reverses the effect. This result has major implications in characterizing how single species survive in the gut, what niche they occupy, and how they engage with other community members.

The antimicrobial activity of zinc against group B Streptococcus is strain-dependent across diverse sequence types, capsular serotypes, and invasive versus colonizing isolates.

BACKGROUND: Streptococcus agalactiae or Group B Streptococcus (GBS) is an encapsulated gram-positive bacterial pathobiont that commonly colonizes the lower gastrointestinal tract and reproductive tract of human hosts. This bacterium can infect the gravid reproductive tract and cause invasive infections of pregnant patients and neonates. Upon colonizing the reproductive tract, the bacterial cell is presented with numerous nutritional challenges imposed by the host. One strategy employed by the host innate immune system is intoxication of bacterial invaders with certain transition metals such as zinc. METHODOLOGY: Previous work has demonstrated that GBS must employ elegant strategies to circumnavigate zinc stress in order to survive in the vertebrate host. We assessed 30 strains of GBS from diverse isolation sources, capsular serotypes, and sequence types for susceptibility or resistance to zinc intoxication. RESULTS: Invasive strains, such as those isolated from early onset disease manifestations of GBS infection were significantly less susceptible to zinc toxicity than colonizing strains isolated from rectovaginal swabs of pregnant patients. Additionally, capsular type III (cpsIII) strains and the ST-17 and ST-19 strains exhibited the greatest resilience to zinc stress, whereas ST-1 and ST-12 strains as well as those possessing capsular type Ib (cpsIb) were more sensitive to zinc intoxication. Thus, this study demonstrates that the transition metal zinc possesses antimicrobial properties against a wide range of GBS strains, with isolation source, capsular serotype, and sequence type contributing to susceptibility or resistance to zinc stress.

Distinct Group B Streptococcus Sequence and Capsule Types Differentially Impact Macrophage Stress and Inflammatory Signaling Responses.

Group B Streptococcus (GBS) is an opportunistic bacterial pathogen that can contribute to the induction of preterm birth in colonized pregnant women and to severe neonatal disease. Many questions regarding the mechanisms that drive GBS-associated pathogenesis remain unanswered, and it is not yet clear why virulence has been observed to vary so extensively across GBS strains. Previously, we demonstrated that GBS strains of different sequence types (STs) and capsule (CPS) types induce different cytokine profiles in infected THP-1 macrophage-like cells. Here, we expanded on these studies by utilizing the same set of genetically diverse GBS isolates to assess ST and CPS-specific differences in upstream cell death and inflammatory signaling pathways. Our results demonstrate that particularly virulent STs and CPS types, such as the ST-17 and CPS III groups, induce enhanced Jun-N-terminal protein kinase (JNK) and NF-κB pathway activation following GBS infection of macrophages compared with other ST or CPS groups. Additionally, we found that ST-17, CPS III, and CPS V GBS strains induce the greatest levels of macrophage cell death during infection and exhibit a more pronounced ability to be internalized and to survive in macrophages following phagocytosis. These data provide further support for the hypothesis that variable host innate immune responses to GBS, which significantly impact pathogenesis, stem in part from genotypic and phenotypic differences among GBS isolates. These and similar studies may inform the development of improved diagnostic, preventive, or therapeutic strategies targeting invasive GBS infections.

Antibiotic Susceptibility Profiles and Frequency of Resistance Genes in Clinical Shiga Toxin-Producing Escherichia coli Isolates from Michigan over a 14-Year Period.

Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen that contributes to over 250,000 infections in the United States each year. Because antibiotics are not recommended for STEC infections, resistance in STEC has not been widely researched despite an increased likelihood for the transfer of resistance genes from STEC to opportunistic pathogens residing within the same microbial community. From 2001 to 2014, 969 STEC isolates were collected from Michigan patients. Antibiotic susceptibility profiles to clinically relevant antibiotics were determined using disc diffusion, while epidemiological data were used to identify factors associated with resistance. Whole-genome sequencing was used for serotyping, examining genetic relatedness, and identifying genetic determinants and mechanisms of resistance in the non-O157 isolates. Increasing frequencies of resistance to at least one antibiotic were observed over the 14 years (P = 0.01). While the non-O157 serogroups were more commonly resistant than O157 (odds ratio, 2.4; 95% confidence interval,1.43 to 4.05), the frequency of ampicillin resistance among O157 isolates was significantly higher in Michigan than the national average (P = 0.03). Genomic analysis of 321 non-O157 isolates uncovered 32 distinct antibiotic resistance genes (ARGs). Although mutations in genes encoding resistance to ciprofloxacin and ampicillin were detected in four isolates, most of the horizontally acquired ARGs conferred resistance to aminoglycosides, ß-lactams, sulfonamides, and/or tetracycline. This study provides insight into the mechanisms of resistance in a large collection of clinical non-O157 STEC isolates and demonstrates that antibiotic resistance among all STEC serogroups has increased over time, prompting the need for enhanced surveillance.

Antibacterial and Anti-biofilm Activity of the Human Breast Milk Glycoprotein Lactoferrin against Group B Streptococcus.

Group B Streptococcus (GBS) is an encapsulated Gram-positive human pathogen that causes invasive infections in pregnant hosts and neonates, as well as immunocompromised individuals. Colonization of the human host requires the ability to adhere to mucosal surfaces and circumnavigate the nutritional challenges and antimicrobial defenses associated with the innate immune response. Biofilm formation is a critical process to facilitate GBS survival and establishment of a replicative niche in the vertebrate host. Previous work has shown that the host responds to GBS infection by producing the innate antimicrobial glycoprotein lactoferrin, which has been implicated in repressing bacterial growth and biofilm formation. Additionally, lactoferrin is highly abundant in human breast milk and could serve a protective role against invasive microbial pathogens. This study demonstrates that human breast milk lactoferrin has antimicrobial and anti-biofilm activity against GBS and inhibits its adherence to human gestational membranes. Together, these results indicate that human milk lactoferrin could be used as a prebiotic chemotherapeutic strategy to limit the impact of bacterial adherence and biofilm formation on GBS-associated disease outcomes.

Analysis of virulence phenotypes and antibiotic resistance in clinical strains of Acinetobacter baumannii isolated in Nashville, Tennessee.

BACKGROUND: Acinetobacter baumannii is a gram-negative bacterium which causes opportunistic infections in immunocompromised hosts. Genome plasticity has given rise to a wide range of strain variation with respect to antimicrobial resistance profiles and expression of virulence factors which lead to altered phenotypes associated with pathogenesis. The purpose of this study was to analyze clinical strains of A. baumannii for phenotypic variation that might correlate with virulence phenotypes, antimicrobial resistance patterns, or strain isolation source. We hypothesized that individual strain virulence phenotypes might be associated with anatomical site of isolation or alterations in susceptibility to antimicrobial interventions. METHODOLOGY: A cohort of 17 clinical isolates of A. baumannii isolated from diverse anatomical sites were evaluated to ascertain phenotypic patterns including biofilm formation, hemolysis, motility, and antimicrobial resistance. Antibiotic susceptibility/resistance to ampicillin-sulbactam, amikacin, ceftriaxone, ceftazidime, cefotaxime, ciprofloxacin, cefepime, gentamicin, levofloxacin, meropenem, piperacillin, trimethoprim-sulfamethoxazole, ticarcillin- K clavulanate, tetracyclin, and tobramycin was determined. RESULTS: Antibiotic resistance was prevalent in many strains including resistance to ampicillin-sulbactam, amikacin, ceftriaxone, ceftazidime, cefotaxime, ciprofloxacin, cefepime, gentamicin, levofloxacin, meropenem, piperacillin, trimethoprim-sulfamethoxazole, ticarcillin- K clavulanate, tetracyclin, and tobramycin. All strains tested induced hemolysis on agar plate detection assays. Wound-isolated strains of A. baumannii exhibited higher motility than strains isolated from blood, urine or Foley catheter, or sputum/bronchial wash. A. baumannii strains isolated from patient blood samples formed significantly more biofilm than isolates from wounds, sputum or bronchial wash samples. An inverse relationship between motility and biofilm formation was observed in the cohort of 17 clinical isolates of A. baumannii tested in this study. Motility was also inversely correlated with induction of hemolysis. An inverse correlation was observed between hemolysis and resistance to ticarcillin-k clavulanate, meropenem, and piperacillin. An inverse correlation was also observed between motility and resistance to ampicillin-sulbactam, ceftriaxone, ceftoxamine, ceftazidime, ciprofloxacin, or levofloxacin. CONCLUSIONS: Strain dependent variations in biofilm and motility are associated with anatomical site of isolation. Biofilm and hemolysis production both have an inverse association with motility in the cohort of strains utilized in this study, and motility and hemolysis were inversely correlated with resistance to numerous antibiotics.

Population Gene Introgression and High Genome Plasticity for the Zoonotic Pathogen Streptococcus agalactiae.

The influence that bacterial adaptation (or niche partitioning) within species has on gene spillover and transmission among bacterial populations occupying different niches is not well understood. Streptococcus agalactiae is an important bacterial pathogen that has a taxonomically diverse host range making it an excellent model system to study these processes. Here, we analyze a global set of 901 genome sequences from nine diverse host species to advance our understanding of these processes. Bayesian clustering analysis delineated 12 major populations that closely aligned with niches. Comparative genomics revealed extensive gene gain/loss among populations and a large pan genome of 9,527 genes, which remained open and was strongly partitioned among niches. As a result, the biochemical characteristics of 11 populations were highly distinctive (significantly enriched). Positive selection was detected and biochemical characteristics of the dispensable genes under selection were enriched in ten populations. Despite the strong gene partitioning, phylogenomics detected gene spillover. In particular, tetracycline resistance (which likely evolved in the human-associated population) from humans to bovine, canines, seals, and fish, demonstrating how a gene selected in one host can ultimately be transmitted into another, and biased transmission from humans to bovines was confirmed with a Bayesian migration analysis. Our findings show high bacterial genome plasticity acting in balance with selection pressure from distinct functional requirements of niches that is associated with an extensive and highly partitioned dispensable genome, likely facilitating continued and expansive adaptation.

Genetic and Phenotypic Factors Associated with Persistent Shedding of Shiga Toxin-Producing Escherichia coli by Beef Cattle.

Shiga toxin-producing Escherichia coli (STEC) is a leading cause of foodborne infections. Cattle are an important STEC reservoir, although little is known about specific pathogen traits that impact persistence in the farm environment. Hence, we sought to evaluate STEC isolates recovered from beef cattle in a single herd in Michigan. To do this, we collected fecal grabs from 26 cattle and resampled 13 of these animals at 3 additional visits over a 3-month period. In all, 66 STEC isolates were recovered for genomics and biofilm quantification using crystal violet assays. The STEC population was diverse, representing seven serotypes, including O157:H7, O26:H11, and O103:H2, which are commonly associated with human infections. Although a core genome analysis of 2,933 genes grouped isolates into clusters based on serogroups, some isolates within each cluster had variable biofilm levels and virulence gene profiles. Most (77.8%; n = 49) isolates harbored stx2a, while 38 (57.5%) isolates formed strong biofilms. Isolates belonging to the predominant serogroup O6 (n = 36; 54.5%) were more likely to form strong biofilms, persistently colonize multiple cattle, and be acquired over time. A high-quality single nucleotide polymorphism (SNP) analysis of 33 O6 isolates detected between 0 and 13 single nucleotide polymorphism (SNP) differences between strains, indicating that highly similar strain types were persisting in this herd. Similar findings were observed for other persistent serogroups, although key genes were found to differ among strong and weak biofilm producers. Together, these data highlight the diversity and persistent nature of some STEC types in this important food animal reservoir.IMPORTANCE Food animal reservoirs contribute to Shiga toxin-producing Escherichia coli (STEC) evolution via the acquisition of horizontally acquired elements like Shiga toxin bacteriophages that enhance pathogenicity. In cattle, persistent fecal shedding of STEC contributes to contamination of beef and dairy products and to crops being exposed to contaminated water systems. Hence, identifying factors important for STEC persistence is critical. This longitudinal study enhances our understanding of the genetic diversity of STEC types circulating in a cattle herd and identifies genotypic and phenotypic traits associated with persistence. Key findings demonstrate that multiple STEC types readily persist in and are transmitted across cattle in a shared environment. These dynamics also enhance the persistence of virulence genes that can be transferred between bacterial hosts, resulting in the emergence of novel STEC strain types. Understanding how pathogens persist and diversify in reservoirs is important for guiding new preharvest prevention strategies aimed at reducing foodborne transmission to humans.