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Raman optical spectroscopy promises label-free bacterial detection, identification, and antibiotic susceptibility testing in a single step. However, achieving clinically relevant speeds and accuracies remains challenging due to weak Raman signal from bacterial cells and numerous bacterial species and phenotypes. Here we generate an extensive dataset of bacterial Raman spectra and apply deep learning approaches to accurately identify 30 common bacterial pathogens. Even on low signal-to-noise spectra, we achieve average isolate-level accuracies exceeding 82% and antibiotic treatment identification accuracies of 97.0±0.3%. We also show that this approach distinguishes between methicillin-resistant and -susceptible isolates of Staphylococcus aureus (MRSA and MSSA) with 89±0.1% accuracy. We validate our results on clinical isolates from 50 patients. Using just 10 bacterial spectra from each patient isolate, we achieve treatment identification accuracies of 99.7%. Our approach has potential for culture-free pathogen identification and antibiotic susceptibility testing, and could be readily extended for diagnostics on blood, urine, and sputum.
Bacterial infections are a leading cause of death in both developed and developing nations, taking >6.7 million lives each year1,2. These infections are also costly to treat, accounting for 8.7% of annual healthcare spending, or $33 billion, in the United States alone3. Current diagnostic methods require sample culturing to detect and identify the bacteria and its antibiotic susceptibility, a slow process that can take days even in state-of-the-art labs4,5. Broad spectrum antibiotics are often prescribed while waiting for culture results6, and according to the Centers for Disease Control and Prevention, over 30% of patients are treated unnecessarily7. New methods for rapid, culture-free diagnosis of bacterial infections are needed to enable earlier prescription of targeted antibiotics and help mitigate antimicrobial resistance.
Beyond empiric first choice antibiotics, clinicians also conduct antibiotic susceptibility tests to determine bacterial responses to drugs. As a step toward a culture-free antibiotic susceptibility test using Raman spectroscopy, we train a binary CNN classifier to differentiate between methicillin-resistant and -susceptible isolates of S. aureus. This model achieves 89.1±0.1% identification accuracy (Fig. 3a). Because the consequences for misdiagnosing MRSA as MSSA are often more severe than the reverse misdiagnosis, the binary decision can be tuned for higher sensitivity (low false negative rate), as shown in the receiver operating characteristic (ROC) curve in Fig. 3b (dotted line denotes performance of random guessing). The area under the curve (AUC) is 0.953, meaning that a randomly selected positive example (i.e., Raman sample from patient with MRSA) will be predicted to be more likely to be MRSA than a randomly selected negative example (i.e., sample from patient with MSSA) with probability 0.953.
Compared to other culture-free methods45 including single-cell sequencing46,47,48,49 and fluorescence or magnetic tagging50, Raman spectroscopy has the unique potential to be a technique for identifying phenotypes that does not require specially designed labels, allowing for easy generalizability to new strains.
Slaves were considered property under Roman law and had no legal personhood. Most slaves would never be freed. Unlike Roman citizens, they could be subjected to corporal punishment, sexual exploitation (prostitutes were often slaves), torture and summary execution. Over time, however, slaves gained increased legal protection, including the right to file complaints against their masters.
The Twelve Tables, Rome's oldest legal code, has brief references to slavery, indicating that the institution was of long standing. In the tripartite division of law by the jurist Ulpian (2nd century AD), slavery was an aspect of the ius gentium, the customary international law held in common among all peoples (gentes). The "law of nations" was neither considered natural law, thought to exist in nature and govern animals as well as humans, nor civil law, belonging to the emerging bodies of laws specific to a people in Western societies. All human beings are born free (liberi) under natural law, but slavery was held to be a practice common to all nations, who might then have specific civil laws pertaining to slaves. In ancient warfare, the victor had the right under the ius gentium to enslave a defeated population; however, if a settlement had been reached through diplomatic negotiations or formal surrender, the people were by custom to be spared violence and enslavement. The ius gentium was not a legal code, and any force it had depended on "reasoned compliance with standards of international conduct".
Vernae (singular verna) were slaves born within a household (familia) or on a family farm or agricultural estate (villa). There was a stronger social obligation to care for vernae, whose epitaphs sometimes identify them as such, and at times they would have been the children of free males of the household. The general Latin word for slave was servus.
During the period of Roman imperial expansion, the increase in wealth amongst the Roman elite and the substantial growth of slavery transformed the economy. Although the economy was dependent on slavery, Rome was not the most slave-dependent culture in history. Among the Spartans, for instance, the slave class of helots outnumbered the free by about seven to one, according to Herodotus. In any case, the overall role of slavery in Roman economy is a discussed issue among scholars.
Delos in the eastern Mediterranean was made a free port in 166 BC and became one of the main market venues for slaves. Multitudes of slaves who found their way to Italy were purchased by wealthy landowners in need of large numbers of slaves to labour on their estates. Historian Keith Hopkins noted that it was land investment and agricultural production which generated great wealth in Italy, and considered that Rome's military conquests and the subsequent introduction of vast wealth and slaves into Italy had effects comparable to widespread and rapid technological innovations.
Nexum was a debt bondage contract in the early Roman Republic. Within the Roman legal system, it was a form of mancipatio. Though the terms of the contract would vary, essentially a free man pledged himself as a bond slave (nexus) as surety for a loan. He might also hand over his son as collateral. Although the bondsman could expect to face humiliation and some abuse, as a legal citizen he was supposed to be exempt from corporal punishment. Nexum was abolished by the Lex Poetelia Papiria in 326 BC, in part to prevent abuses to the physical integrity of citizens who had fallen into debt bondage.
Roman historians illuminated the abolition of nexum with a traditional story that varied in its particulars; basically, a nexus who was a handsome but upstanding youth suffered sexual harassment by the holder of the debt. In one version, the youth had gone into debt to pay for his father's funeral; in others, he had been handed over by his father. In all versions, he is presented as a model of virtue. Historical or not, the cautionary tale highlighted the incongruities of subjecting one free citizen to another's use, and the legal response was aimed at establishing the citizen's right to liberty (libertas), as distinguished from the slave or social outcast (infamis).
Epitaphs record at least 55 different jobs a household slave might have, including barber, butler, cook, hairdresser, handmaid (ancilla), wash their master's clothes, wet nurse or nursery attendant, teacher, secretary, seamstress, accountant, and physician. A large elite household (a domus in town, or a villa in the countryside) might be supported by a staff of hundreds. The living conditions of slaves attached to a domus (the familia urbana), while inferior to those of the free persons they lived with, were sometimes superior to that of many free urban poor in Rome. Household slaves likely enjoyed the highest standard of living among Roman slaves, next to publicly owned slaves, who were not subject to the whims of a single master. Imperial slaves were those attached to the emperor's household, the familia Caesaris.
Slaves numbering in the tens of thousands were condemned to work in the mines or quarries, where conditions were notoriously brutal. Damnati in metallum ("those condemned to the mine") were convicts who lost their freedom as citizens (libertas), forfeited their property (bona) to the state, and became servi poenae, slaves as a legal penalty. Their status under the law was different from that of other slaves; they could not buy their freedom, be sold, or be set free. They were expected to live and die in the mines. Imperial slaves and freedmen (the familia Caesaris) worked in mine administration and management.
Because they had an opportunity to prove their merit, they could acquire a reputation and influence, and were sometimes deemed eligible for manumission. During the Republic, a public slave could be freed by a magistrate's declaration, with the prior authorization of the senate; in the Imperial era, liberty would be granted by the emperor. Municipal public slaves could be freed by the municipal council.
Rome differed from Greek city-states in allowing freed slaves to become citizens. After manumission, a male slave who had belonged to a Roman citizen enjoyed not only passive freedom from ownership, but active political freedom (libertas), including the right to vote. A slave who had acquired libertas was thus a libertus ("freed person", feminine liberta) in relation to his former master, who then became his patron (patronus). As a social class, freed slaves were libertini, though later writers used the terms libertus and libertinus interchangeably. Libertini were not entitled to hold public office or state priesthoods, nor could they achieve senatorial rank. During the early Empire, however, freedmen held key positions in the government bureaucracy, so much so that Hadrian limited their participation by law. Any future children of a freedman would be born free, with full rights of citizenship. 2b1af7f3a8