Ruth N. Zadoks

Genomic analysis of diversity, population structure, virulence, and antimicrobial resistance in Klebsiella pneumoniae, an urgent threat to public health

Significance Klebsiella pneumoniae is rapidly becoming untreatable using last-line antibiotics. It is especially problematic in hospitals, where it causes a range of acute infections. To approach controlling such a bacterium, we first must define what it is and how it varies genetically. Here we have determined the DNA sequence of K. pneumoniae isolates from around the world and present a detailed analysis of these data. We show that there is a wide spectrum of diversity, including variation within shared sequences and gain and loss of whole genes. Using this detailed blueprint, we show that there is an unrecognized association between the possession of specific gene profiles associated with virulence and antibiotic resistance and the differing disease outcomes seen for K. pneumoniae. Klebsiella pneumoniae is now recognized as an urgent threat to human health because of the emergence of multidrug-resistant strains associated with hospital outbreaks and hypervirulent strains associated with severe community-acquired infections. K. pneumoniae is ubiquitous in the environment and can colonize and infect both plants and animals. However, little is known about the population structure of K. pneumoniae, so it is difficult to recognize or understand the emergence of clinically important clones within this highly genetically diverse species. Here we present a detailed genomic framework for K. pneumoniae based on whole-genome sequencing of more than 300 human and animal isolates spanning four continents. Our data provide genome-wide support for the splitting of K. pneumoniae into three distinct species, KpI (K. pneumoniae), KpII (K. quasipneumoniae), and KpIII (K. variicola). Further, for K. pneumoniae (KpI), the entity most frequently associated with human infection, we show the existence of >150 deeply branching lineages including numerous multidrug-resistant or hypervirulent clones. We show K. pneumoniae has a large accessory genome approaching 30,000 protein-coding genes, including a number of virulence functions that are significantly associated with invasive community-acquired disease in humans. In our dataset, antimicrobial resistance genes were common among human carriage isolates and hospital-acquired infections, which generally lacked the genes associated with invasive disease. The convergence of virulence and resistance genes potentially could lead to the emergence of untreatable invasive K. pneumoniae infections; our data provide the whole-genome framework against which to track the emergence of such threats.

Listeria monocytogenes isolates from foods and humans form distinct but overlapping populations

ABSTRACT A total of 502 Listeria monocytogenes isolates from food and 492 from humans were subtyped by EcoRI ribotyping and PCR-restriction fragment length polymorphism analysis of the virulence gene hly. Isolates were further classified into genetic lineages based on subtyping results. Food isolates were obtained through a survey of selected ready-to-eat food products in Maryland and California in 2000 and 2001. Human isolates comprised 42 isolates from invasive listeriosis cases reported in Maryland and California during 2000 and 2001 as well as an additional 450 isolates from cases that had occurred throughout the United States, predominantly from 1997 to 2001. Assignment of isolates to lineages and to the majority of L. monocytogenes subtypes was significantly associated with the isolate source (food or human), although most subtypes and lineages included both human and food isolates. Some subtypes were also significantly associated with isolation from specific food types. Tissue culture plaque assay characterization of the 42 human isolates from Maryland and California and of 91 representative food isolates revealed significantly higher average infectivity and cell-to-cell spread for the human isolates, further supporting the hypothesis that food and human isolates form distinct populations. Combined analysis of subtype and cytopathogenicity data showed that strains classified into specific ribotypes previously linked to multiple human listeriosis outbreaks, as well as those classified into lineage I, are more common among human cases and generate larger plaques than other subtypes, suggesting that these subtypes may represent particularly virulent clonal groups. These data will provide a framework for prediction of the public health risk associated with specific L. monocytogenes subtypes.

Host-response patterns of intramammary infections in dairy cows

Many different bacterial species have the ability to cause an infection of the bovine mammary gland and the host response to these infections is what we recognize as mastitis. In this review we evaluate the pathogen specific response to the three main bacterial species causing bovine mastitis: Escherichia coli, Streptococcus uberis and Staphylococcus aureus. In this paper we will review the bacterial growth patterns, host immune response and clinical response that results from the intramammary infections. Clear differences in bacterial growth pattern are shown between bacterial species. The dominant pattern in E. coli infections is a short duration high bacteria count infection, in S. aureus this is more commonly a persistent infection with relative low bacteria counts and in S. uberis a long duration high bacteria count infection is often observed. The host immune response differs significantly depending on the invading bacterial species. The underlying reasons for the differences and the resulting host response are described. Finally we discuss the clinical response pattern for each of the three bacterial species. The largest contrast is between E. coli and S. aureus where a larger proportion of E. coli infections cause potentially severe clinical symptoms, whereas the majority of S. aureus infections go clinically unnoticed. The relevance of fully understanding the bovine host response to intramammary infection is discussed, some major gaps in our knowledge are highlighted and directions for future research are indicated.

Multilocus Sequence Typing of Intercontinental Bovine Staphylococcus aureus Isolates

ABSTRACT A total of 258 bovine-associated Staphylococcus aureus isolates from the United States, Chile, and the United Kingdom, plus the reference isolate S. aureus Newbould 305 (NCIMB 702892), were analyzed by multilocus sequence typing (MLST). A collection of previously characterized United Kingdom isolates were also included in the analysis. The results demonstrated that MLST is suitable for the differentiation of bovine S. aureus isolates from various sites (milk, teat skin, milking machine unit liners, hands, and bedding) and countries. The theory of the host specificity of S. aureus is supported by the detection of a previously undescribed clonal complex that comprised 87.4% of the isolates studied, with representatives from all geographic locations investigated. This suggests that a single clonal group has achieved a widespread distribution and is responsible for the majority of infections. Some sequence types (STs; ST25, ST115, ST124, and ST126) demonstrated site specificity, as they were significantly (P < 0.05) associated with milk or teat skin.

Molecular Epidemiology of Mastitis Pathogens of Dairy Cattle and Comparative Relevance to Humans

Mastitis, inflammation of the mammary gland, can be caused by a wide range of organisms, including gram-negative and gram-positive bacteria, mycoplasmas and algae. Many microbial species that are common causes of bovine mastitis, such as Escherichia coli, Klebsiella pneumoniae, Streptococcus agalactiae and Staphylococcus aureus also occur as commensals or pathogens of humans whereas other causative species, such as Streptococcus uberis, Streptococcus dysgalactiae subsp. dysgalactiae or Staphylococcus chromogenes, are almost exclusively found in animals. A wide range of molecular typing methods have been used in the past two decades to investigate the epidemiology of bovine mastitis at the subspecies level. These include comparative typing methods that are based on electrophoretic banding patterns, library typing methods that are based on the sequence of selected genes, virulence gene arrays and whole genome sequencing projects. The strain distribution of mastitis pathogens has been investigated within individual animals and across animals, herds, countries and host species, with consideration of the mammary gland, other animal or human body sites, and environmental sources. Molecular epidemiological studies have contributed considerably to our understanding of sources, transmission routes, and prognosis for many bovine mastitis pathogens and to our understanding of mechanisms of host-adaptation and disease causation. In this review, we summarize knowledge gleaned from two decades of molecular epidemiological studies of mastitis pathogens in dairy cattle and discuss aspects of comparative relevance to human medicine.

Whole genome sequencing identifies zoonotic transmission of MRSA isolates with the novel mecA homologue mecC

Several methicillin‐resistant Staphylococcus aureus (MRSA) lineages that carry a novel mecA homologue (mecC) have recently been described in livestock and humans. In Denmark, two independent human cases of mecC‐MRSA infection have been linked to a livestock reservoir. We investigated the molecular epidemiology of the associated MRSA isolates using whole genome sequencing (WGS). Single nucleotide polymorphisms (SNP) were defined and compared to a reference genome to place the isolates into a phylogenetic context. Phylogenetic analysis revealed two distinct farm‐specific clusters comprising isolates from the human case and their own livestock, whereas human and animal isolates from the same farm only differed by a small number of SNPs, which supports the likelihood of zoonotic transmission. Further analyses identified a number of genes and mutations that may be associated with host interaction and virulence. This study demonstrates that mecC‐MRSA ST130 isolates are capable of transmission between animals and humans, and underscores the potential of WGS in epidemiological investigations and source tracking of bacterial infections.

Comparison of Staphylococcus aureus Isolates from Bovine and Human Skin, Milking Equipment, and Bovine Milk by Phage Typing, Pulsed-Field Gel Electrophoresis, and Binary Typing

ABSTRACT Staphylococcus aureus isolates (n = 225) from bovine teat skin, human skin, milking equipment, and bovine milk were fingerprinted by pulsed-field gel electrophoresis (PFGE). Strains were compared to assess the role of skin and milking equipment as sources of S. aureus mastitis. PFGE of SmaI-digested genomic DNA identified 24 main types and 17 subtypes among isolates from 43 herds and discriminated between isolates from bovine teat skin and milk. Earlier, phage typing (L. K. Fox, M. Gershmann, D. D. Hancock, and C. T. Hutton, Cornell Vet. 81:183-193, 1991) had failed to discriminate between isolates from skin and milk. Skin isolates from humans belonged to the same pulsotypes as skin isolates from cows. Milking equipment harbored strains from skin as well as strains from milk. We conclude that S. aureus strains from skin and from milk can both be transmitted via the milking machine, but that skin strains are not an important source of intramammary S. aureus infections in dairy cows. A subset of 142 isolates was characterized by binary typing with DNA probes developed for typing of human S. aureus. Typeability and overall concordance with epidemiological data were lower for binary typing than for PFGE while discriminatory powers were similar. Within several PFGE types, binary typing discriminated between main types and subtypes and between isolates from different herds or sources. Thus, binary typing is not suitable as replacement for PFGE but may be useful in combination with PFGE to refine strain differentiation.

Occurrence of Mycobacterium avium subspecies paratuberculosis across host species and European countries with evidence for transmission between wildlife and domestic ruminants

BackgroundMycobacterium avium subspecies paratuberculosis (Map) causes an infectious chronic enteritis (paratuberculosis or Johnes disease) principally of ruminants. The epidemiology of Map is poorly understood, particularly with respect to the role of wildlife reservoirs and the controversial issue of zoonotic potential (Crohns disease). Genotypic discrimination of Map isolates is pivotal to descriptive epidemiology and resolving these issues. This study was undertaken to determine the genetic diversity of Map, enhance our understanding of the host range and distribution and assess the potential for interspecies transmission.Results164 Map isolates from seven European countries representing 19 different host species were genotyped by standardized IS900 - restriction fragment length polymorphism (IS900-RFLP), pulsed-field gel electrophoresis (PFGE), amplified fragment length polymorphisms (AFLP) and mycobacterial interspersed repeat unit-variable number tandem repeat (MIRU-VNTR) analyses. Six PstI and 17 BstEII IS900-RFLP, 31 multiplex [SnaBI-SpeI] PFGE profiles and 23 MIRU-VNTR profiles were detected. AFLP gave insufficient discrimination of isolates for meaningful genetic analysis. Point estimates for Simpsons index of diversity calculated for the individual typing techniques were in the range of 0.636 to 0.664 but a combination of all three methods increased the discriminating power to 0.879, sufficient for investigating transmission dynamics. Two predominant strain types were detected across Europe with all three typing techniques. Evidence for interspecies transmission between wildlife and domestic ruminants on the same property was demonstrated in four cases, between wildlife species on the same property in two cases and between different species of domestic livestock on one property.ConclusionThe results of this study showed that it is necessary to use multiple genotyping techniques targeting different sources of genetic variation to obtain the level of discrimination necessary to investigate transmission dynamics and trace the source of Map infections. Furthermore, the combination of genotyping techniques may depend on the geographical location of the population to be tested. Identical genotypes were obtained from Map isolated from different host species co-habiting on the same property strongly suggesting that interspecies transmission occurs. Interspecies transmission of Map between wildlife species and domestic livestock on the same property provides further evidence to support a role for wildlife reservoirs of infection.

CNS mastitis: Nothing to worry about?

In this paper, we analyzed a very large field data set on intramammary infections (IMI) and the associated somatic cell count (SCC) in dairy cows. The objective of the study was to analyze the impact of coagulase-negative staphylococci (CNS) IMI on cow SCC, both mean and variability, and on the potential of these infections to have a major impact on the bulk milk SCC (BMSCC). Data and milk samples for bacterial culture were collected by Quality Milk Production Services (QMPS) between 1992 and March of 2007. The QMPS program services dairy farms in New York State and other states in the Northeastern USA and operates in conjunction with Cornell University. Only records from cows where SCC and milk production data were available, and where only one organism was isolated from bacterial cultures of milk samples (or where culture was negative) were used for this analysis. A total of 352,614 records from 4200 whole herd mastitis screening sampling qualified for this study. Within herds an average of 15% (S.D. 12%) of cows sampled were infected with CNS, ranging between 0 and 100%. Average within herd prevalence of cows with a CNS IMI and an SCC over 200,000 cells/ml was 2% (S.D. 4%) with a minimum of 0% and a maximum of 50%. Results of linear mixed models showed three distinct populations of IMI statuses: negative cultures with the lowest SCC; CNS and Corynebacterium bovis with a moderate increase in SCC, and Streptococcus agalactiae, Streptococcus spp. and Staphylococcus aureus showing an important increase in SCC. Surprisingly, milk production was slightly but significantly higher in CNS infected cows compared to culture-negative cows, whereas it was strongly reduced in cows with a major pathogen IMI. The percentage contribution of CNS infections to the BMSCC was 17.9% in herds with a BMSCC less than 200,000 cells/ml. This value decreased to 11.9 and 7.9% in herds with bulk milk SCC between 200,000 and 400,000 and over 400,000 cells/ml, respectively. We concluded that very few herds with milk quality problems would have an important increase in BMSCC that could be mostly attributed to CNS infections. On the other hand, in herds with low BMSCC, CNS infections may be an important contributor to the total number of somatic cells in the bulk milk.

Clinical, epidemiological and molecular characteristics of Streptococcus uberis infections in dairy herds.

A longitudinal observational study (18 months) was carried out in two Dutch dairy herds to explore clinical, epidemiological and molecular characteristics of Streptococcus uberis mastitis. Infections (n = 84) were detected in 70 quarters of 46 cows. Bacterial isolates were characterized at strain level by random amplified polymorphic DNA (RAPD) fingerprinting. Persistent infections were usually attributable to one strain, while recurrent infections could be caused by different strains. When multiple quarters of a cow were infected, infections were mostly caused by one strain. In each herd, multiple strains were identified yet one strain predominated. The majority of all infections were subclinical, and infections attributed to predominant strains were more chronic than infections attributed to other strains. Epidemiological and molecular data suggest infection from environmental sources with a variety of S. uberis strains as well as within-cow and between-cow transmission of a limited number of S. uberis strains, with possible transfer of bacteria via the milking machine.